Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp, and Listeria monocytogenes DNA in milk / [Padronização de um método rápido de PCR quadriplex para a detecção simultânea de DNA de bovino, bubalino, Salmonella spp. e Listeria monocytogenes em leite]

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.

Padronização de uma PCR para a autenticação do Salmo salar em pratos da culinária japonesa / Standardization of a PCR for the authentication of Salmo salar in dishes of Japanese cuisine

O objetivo deste trabalho foi padronizar uma PCR para a detecção do Salmo salar, a qual possa ser usada na autenticação do salmão utilizado em pratos da culinária japonesa e do pescado comercializado in natura. Para isso, dois lotes de sushi foram produzidos experimentalmente. Além disso, foram visitados 38 estabelecimentos que comercializam comida japonesa e 10 peixarias na região metropolitana de Belém, visando à coleta do sushi, do temaki e do pescado pertencente à espécie Salmo salar. Os dados demonstraram que a técnica foi eficiente para a autenticação de Salmo salar, visto que a espécie foi detectada tanto nas amostras de sushis preparados experimentalmente quanto nas alíquotas de pescados isolados, utilizados para a preparação do sushi. Em contrapartida, a espécie Salmo trutta não foi detectada nas amostras de sushis preparados com esta espécie nem nas alíquotas de pescado isolado. Além disso, foi possível a confirmação da utilização da espécie Salmo salar no preparo das amostras de sushi, temaki e de pescado. Portanto, concluiu-se que a técnica foi capaz de amplificar o DNA da referida espécie e não gerou identificação inespecífica quando a espécie Salmo trutta foi analisada, podendo ser uma ferramenta adequada para a autenticação do Salmo salar.(AU)

The objective of this work was to standardize a PCR for the detection of Salmo salar, which can be used in the authentication of salmon used in Japanese dishes and fish commercialized in natura. For this, two batches of sushi were produced experimentally. In addition, 38 establishments that sell Japanese food and 10 fishmongers in the metropolitan area of Belém were visited, aiming to collect sushi, temaki and fish belonging to the species Salmo salar. The data demonstrated that the technique was efficient for the authentication of Salmo salar, since the species was detected in both the experimentally prepared sushi samples and the isolated fish aliquots used for the preparation of sushi. In contrast, the species Salmo trutta was not detected in the sushi samples prepared with this species nor in the isolated fish aliquots. In addition, it was possible to confirm the use of the Salmo salar species in the preparation of sushi, temaki and fish samples. Therefore, it was concluded that the technique was able to amplify the DNA of this species and did not generate nonspecific identification when the species Salmo trutta was analyzed, being able to be a suitable tool for the authentication of Salmo salar.(AU)

Saccharomyces boulardii modulates and improves the immune response to Bovine Herpesvirus type 5 Vaccine / Saccharomyces boulardii modula e melhora a resposta imune de vacina contra o Herpes bovino tipo 5

There have been significant efforts towards the development of more efficient vaccines for animal health. A strategy that may be used to improve vaccine efficacy is the use of probiotics to enhance the immune response of the host, leading to increased immunogenicity of antigen preparations. Bovine herpesvirus 5 (BoHV-5) is an example of an important animal pathogen for which vaccines have provided only limited protection. In this study, we examined the use of the probiotic Saccharomyces boulardii (Sb) as a potential adjuvant to improve vaccine efficiency. We found that the supplemented animals exhibited an enhanced systemic IgG antibody response toward a Th1 response in favor of IgG2a and increased mRNA expression levels of the cytokines IFN-y, IL-12, IL-17 and IL-10 in the spleen. These results suggest that Sb supplementation may provide a promising means for improving the efficiency of vaccines, particularly those that rely on a cell-mediated immune response.(AU)

Esforços significativos têm sido realizados para o desenvolvimento de vacinas mais eficientes em saúde animal. Uma estratégia que pode ser usado para melhorar a eficácia da vacina é o uso de probióticos para melhorar a resposta imune do hospedeiro, conduzindo ao aumento da imunogenicidade de preparações de antígenos. Herpesvírus bovino 5 (BoHV-5) é um exemplo de um importante patógeno animal para os quais vacinas têm fornecido apenas uma protecção limitada. Neste estudo, examinou-se o uso do probiótico Saccharomyces boulardii (Sb) como um adjuvante potencial para melhorar a eficiência da vacina. Verificou-se que os animais suplementados apresentaram uma produção de anticorpos IgG superior e com desvio para Th1 em favor de IgG2, além do aumento dos níveis de expressão de mRNA para as citocinas IFN-γ, IL-12, IL-17 e IL-10. Esses resultados sugerem que a suplementação de Sb pode fornecer um meio promissor para melhorar a eficiência de vacinas, particularmente aquelas que dependem de uma resposta imune mediada por células.(AU)

The use of quorum sensing to improve vaccine immune response.

Enterotoxigenic Escherichia coli (ETEC) infection is an important cause of diarrhea in both newborn and post-weaning pigs, it is also responsible for economic losses on farms worldwide. Vaccines that use ETEC virulence factors have been well documented, and several vaccines containing inactivated bacteria with protective antigens, or purified (isolated) antigens are available on the market. Vaccination of pregnant sows is widely seen as an effective strategy for the control of the disease. Yet these vaccines very often do not lead to efficient protection. In this study, we produced an ETEC bacterin with the use of quorum sensing (QS), and observed a significant expression of F4 adhesin, and heat-labile toxin (LT) in the cultures when compared to the controls. Mice, and pigs vaccinated with the QS bacterin demonstrated higher antibody titers against these antigens when compared with commercial and control bacterin. Our results suggest that the system might bring promising improvements in ETEC bacterin efficacy.

Avaliação microbiológica de queijo colonial produzido na cidade de Três Passos, RS / Microbiological evaluation of produced cheese in the city of Três Passos, RS

Vinte e cinco amostras de queijo colonial, oriundas de cinco estabelecimentos da cidade de Três Passos, RS, foram submetidas às seguintes análises microbiológicas: contagens de bactérias mesófilas, bactérias psicrotróficas, coliformes totais, coliformes fecais e pesquisa de Salmonella. As médias das contagens de microorganismos mesófilos e psicrotróficos foram, na propriedade A, 4,3 x 10 UFC/g e 1,4 x 10 UFC/g; na B, 3,7 x 10 UFC/g e 4,4 x 10 UFC/g; na C, 2,5 x 10 UFC/g e 2,8 x 10 UFC/g; na D, 1,4 x 10 UFC/ g e 4,0 x 10 UFC/g e, na E, 1,5 x 10 UFC/g e 6,7 x 10 UFC/g, respectivamente. As contagens de coliformes totais nas propriedades A, B, C e D foram > 110 NMP/g e na propriedade E 88,9 NMP. Os resultados das contagens coliformes fecais foram 47,5 NMP/g na propriedade A, >110 NMP/g na B,2,1 NMP/g na C, <0,3 NMP/g na D e 10,0 NMP/g na E. A pesquisa de Salmonella apresentou ausência / 25g em todas as amostras. Os resultados revelaram altos índices de microorganismos nos produtos, sugerindo qualidade de matéria prima insatisfatória e/ou condições de produção e armazenamento inadequadas.

Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls.

The amount of non-cell-associated DNA free in blood plasma from pancreatic cancer patients usually exceeds that from healthy donors. We have evaluated the plasma DNA by gel electrophoresis and measured the variation in length of soluble DNA fragments by electron microscopy in plasma from three patients with pancreatic cancer and from three healthy controls. Whereas electrophoresis of nick-translated DNA isolated from plasma obtained from healthy controls showed autoradiographic bands at sizes equivalent to whole-number multiples (1-5x) of nucleosomal DNA (185-200 bp), in the samples obtained from pancreatic cancer patients, stronger ladder patterns appeared. Likewise, strand length distributions of DNA (DNA-SL) in the two groups differ. The DNA-SL distribution data include 2,752 measurements made from cancer patient plasma and 3,291 for control plasma. The shortest DNA-SL measured approximately 30 nm (approximately 88 bp calculated at 0.34 nm/bp) and the largest approximately 28,000 nm (>80,000 bp), with 50% of all lengths measuring between 100 and 900 nm long. The average plasma DNA-SL in controls (311 nm; median, 273 nm) exceeded that in cancer patients (231 nm; median, 185 nm). Small excesses of DNA at approximately 63, approximately 126, approximately 189, approximately 252, and approximately 315 nm, corresponding to small multiples of lengths associated with nucleosomes, were more prominent in the cancer patient plasma than in the healthy control plasma. This study provides evidence indicating differences in non-cell-associated DNA in plasma between cancer patients and healthy controls and indicates that a significant amount of this DNA is probably derived from apoptosis in neoplastic and/or normal cells.

Conformation of Lac repressor tetramer in solution, bound and unbound to operator DNA.

We tested whether the Steitz et al. [(1974) Proc. Natl. Acad. Sci. U.S.A., 71:593-597] model of lactose repressor (LacR) (14 x 6.0 x 4.5 nm) represented the shape of free or operator-bound LacR in solution. The model predicts a 14 nm length for bound LacR. Direct measurement, using Pt-C shadow width standards, was 9.6 +/- 0.2 nm long. Using the Steitz model, we generated a distribution of measurements and converted them into a distribution of shadow widths using gold ball standards. Direct measurement of LacR produced a narrower shadow width distribution with a larger mean size than the Steitz model predicted. Measurement along two orthogonal axes of negatively stained LacR images generated a size distribution, also converted into a shadow width distribution using the gold ball standards. Since the experimental shadow width distribution exactly matched the shadow width distribution derived theoretically from negatively stained LacR, our negative-stained images are representative of LacR's conformation in solution. Approximately 56% of negatively stained LacR had a V-shaped fold around an axis orthogonal to its length, bringing the DNA binding domains of each dimer adjacent. This open end of the V binds single operator DNA. The other 44% of the LacR tetramer is in the extended form with its DNA binding sites at opposite ends. Although the V-shaped conformation has a closed hinge with the dimers associated along a side, the extended open-hinged state remains important since LacR must bind two distant operator sites for full repression. Our measurements predict the normal presence of both conformations in nearly equal amounts, suggesting that both are equally active in repressing the lac operon.

The formation of the gonadal ridge in Xenopus laevis. III. The behaviour of isolated primordial germ cells in vitro.

Previous studies have described the morphology, including the ultrastructure, of primordial germ cells (PGCs), and the cells with which they associate to form the gonadal ridge, in Xenopus laevis. In order to test their capacity for active movement we have studied single, isolated PGCs in vitro. Time-lapse studies of these cells reveal that they are motile, using broad cytoplasmic processes. The fact that these cells are very large and easy to manipulate in vitro makes them an attractive subject of study, particularly with respect to the mechanism of their movement and the surface phenomena which guide them to the site of the gonadal ridge.

Development of Rathke's pouch tissue transplanted into adult hypophysectomized female rats.

Rathke's pouch tissue, from rat embryos of 11-15 days' gestation, was microsurgically removed and transplanted into the hypothalamus of hypophysectomized adult females. The hosts were sacrificed 4 weeks later; brain and target organs were preserved for histological examination. Plasma samples were taken for the radioimmunoassay (RIA) of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The implanted tissue invariably developed along certain lines. Undifferentiated primitive cells were found associated with nervous tissue, dense connective tissue, cartilage and glandular cells. In every age group, some implants became invasive, forming massive growths in the brain. These tumorous properties were principally associated (p less than 0.05) with tissue from 12-day embryos. Pituitary primordia from all ages demonstrated the ability to develop into functional adenohypophyseal tissue. Target organ stimulation indicated a secretion of corticotropin, thyrotropin and somatotropin. FSH and/or LH were detected by RIA in the plasma of 61% of the test animals. We suggest that this system offers unique opportunities for future investigations into the mechanisms that determine whether embryonic epithelial tissue will remain under normal growth control or will become tumorous or even cancerous.