M.R. Souza

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

BackgroundThe use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization.ResultsBacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy.ConclusionLactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.

Effects of lipid sources in the diet of laying hens on the fatty acid profiles of egg yolks

The effects of different dietary lipids on the fatty acid profiles of eggs produced by 20- and 54-wk-old Dekalb laying hens were investigated. The 4 treatments were based on the lipid source added to the diet: soybean oil, sunflower oil, linseed oil, or control (no added oil). The experimental design was a simple random-sample design using a 4 × 2 factorial arrangement (4 treatments and 2 ages). The fatty acid composition of the yolks of eggs produced by the laying hens was analyzed. The fatty acid profiles found in the egg yolks were the same as those provided in each diet. Eggs laid by hens fed the diet containing soybean oil had a large amount of n-6 polyunsaturated fatty acids (PUFA), whereas eggs laid by hens fed the diet containing linseed oil had the highest percentage of n-3 PUFA. A decrease in PUFA deposition in egg yolks was observed as the laying hens got older. Eggs of hens fed the diet containing linseed oil presented an n-6:n-3 ratio of 2.01 in younger chickens and 2.17 in older ones. The trans fat percentages found in the egg yolks of all treatments were very low. It was concluded that the quantity of fatty acids present in the egg yolk may be altered according to the source of lipids in the diets; the addition of linseed oil to the ration of laying hens resulted in the production of n-3-enriched eggs and excellent n-6:n-3 ratios, and the egg yolks had insignificant amounts of trans fat, irrespective of the different lipid sources added to the diets or the age of the chickens.

Qualidade de ovos de consumo submetidos a diferentes condições de armazenamento

The internal quality of 768 commercial white eggs from 40-week-old Hy-Line laying hens was evaluated after storage under different conditions. The eggs were submitted to treatments in a factorial scheme of 2x4x8, being two types of package (using plastic film or not), four periods of storage (zero, five, 10, and 15 days), and eight refrigerated storage times (zero, five, 10, 15, 20, 25, 30 and 35 days). The values of Haugh Unities (HU) and albumen pH were measured. The HU values significantly decreased (P<0.05) during storage mainly at room temperature. Wrapping the eggs packages with plastic film showed efficient in preserving their internal quality since it kept HU higher for a longer storage time. The pH values of the eggs increased during the storage time (P<0.05), independently of both storage temperature and packaging. It was concluded that temperature and time of storage of eggs influenced on the values of HU and wrapping the eggs packages with plastic film improved their internal quality.

Atividade antimicrobiana de bactérias lácticas isoladas de queijo-de-minas artesanal do Serro (MG) frente a microrganismos indicadores

A total of 192 strains of lactic acid bacteria were isolated from five samples of artisanal minas cheese, from Serro region - Minas Gerais State. The results of direct inhibition test indicated that 48 strains (25%) inhibited the in vitro growth of the indicator microorganisms: Staphylococcusaureus and Listeriamonocytogenes. Those 48 strains were tested by the indirect inhibition test, and 10 strains (20.8%) showed a wide antimicrobial spectrum against Salmonellaenteritidisvar. typhimurium, Staphylococcusaureus, Listeria monocytogenes and Listeria innocua. None of the isolated strains was able to inhibit Escherichia coli.

Bioactive amines and quality of egg from Dekalb hens under different storage conditions

The objective of this study was to investigate the possibility of using bioactive amines as an index of quality of fresh and stored eggs. Large white eggs from 24-wk-old Dekalb layers were randomly distributed in 3 groups: (i) 10 freshly laid eggs, (ii) 60 eggs stored at 25 +/- 1 degrees C and 60% RH for 30 d, and (iii) 50 eggs stored at 6 +/- 1 degrees C and 60% RH for 50 d. The eggs were weighed and the internal quality was evaluated by Haugh units (HU), pH of albumen, total solids, total volatile bases, and bioactive amines in the albumen and yolk. The fresh eggs had, on average, 56.85 g, 98.55 HU, albumen pH of 8.02, total solids of 12.17 g/100 g in the albumen and 52.43 g/100 g in the yolk, and absence of volatile bases. None of the 10 amines investigated were detected in the albumen; however, the yolk contained 0.37 mg/kg of spermidine. Throughout storage, there was a significant decrease in the weight of the egg and HU and a significant increase in the pH and in the total solids of the albumen. The decrease in HU and the increase in the total solids of the albumen were faster at 25 +/- 1 degrees C compared with 6 +/- 1 degrees C. At 50 and 30 d of storage at 6 +/- 1 and 25 +/- 1 degrees C, respectively, significant levels of total volatile bases were detected. The levels of spermidine in the yolk increased significantly at the 40th and 15th days of storage at 6 +/- 1 and 25 +/- 1 degrees C, respectively. At these storage times, the presence of putrescine and agmatine was also detected. Therefore, the presence of other amines besides spermidine or spermidine levels higher than 1.0 mg/kg in the yolk could be used as an index of quality of fresh eggs and throughout storage.

Contagem de células somáticas em leite de cabra

This study was carried out to compare different methods used for enumerating somatic cells in cow milk in order to determine which of them would give accurate estimates of somatic cell counts (SCC) in goat milk. Fifteen samples of goat milk were analyzed by direct microscopy, flow citometry using Somacount 300 equipment and Wisconsin Mastitis Test (WMT). The results were, respectively, 6.07 log SCC/ml, 6.08 log SCC/ml and 5.54 log SCC/ml. The SCC mean obtained by Somacount 300 was similar (P>0.05) to that showed by direct microscopy count, while WMT underestimated goat milk SCC.

Qualidade de ovos comerciais submetidos a diferentes condições de armazenamento

Physical-chemical egg qualities of 1440 egg from commercial layer hens of different ages (30 and 60-week-old) submitted to storage under room temperature or refrigeration conditions were evaluated according to the following traits: Haugh Unities (UH); pH of albumen (pH); total solids of albumen and yolk; and weight and percentage of albumen, yolk, and shell. Decrease of inner quality of the evaluated eggs was observed during the experiment, mainly eggs from old-layers, which presented the worst values of AA and HU when stored at room temperature. It was concluded that age, temperature and time of storage of eggs influenced egg quality.

Contagem de Staphylococcus sp. e detecção de enterotoxinas estafilocócicas e toxina da síndrome do choque tóxico em amostras de leite cru refrigerado

In order to count and identify Staphylococcus sp., the detection of the Staphylococcal enterotoxins (SE) and toxic shock toxin syndrome (TSST-1), 80 raw milk samples cooled at 4°C and stored in bulk tanks for 48 hours in different farms from Minas Gerais State were analyzed. Staphylococcus sp. was observed in all samples and the counts varied from 1.0 × 105 to 2.5 × 107 CFU/ml (mean = 5.60 log CFU/ml; sd = 0.53 and CV = 9.46 %). A total of 436 strains of Staphylococcus were isolated and identified as S. aureus, S hyicus, S. epidermidis, S. intermedius, S. cohnii, S. sciuri, S. schleirferi and S. delphini. Strains showing identical biochemical profile, from the same sample, were grouped into a pool and them were induced to produce SE and TSST-1. The detection of toxins was made by the OSP (Optimum Sensivity Plate) method and the cellophane-over-agar technique. It was identified SEA, SEB, SEC, SED and TSST-1 in different percentages. From the 138 formed pools, 91 produced, at least, one or more toxin, including TSST-1. From the enterotoxigenic pools, 24.6% were coagulase positive, while 41.3% were negative. The presence of entorotoxigenic negative coagulase Staphylococcus strains isolated from milk samples is important in relation to public health.

Produção e composição do leite de ovelhas Santa Inês e mestiças Lacaune e Santa Inês e desenvolvimento de seus cordeiros

Na ovinocultura, a raca Santa Ines, amplamente explorada no territorio brasileiro, possui indices de producao compativeis com sistemas europeus de exploracao leiteira (Susin et al., 2005), e o bom desempenho de animais na fase de cria esta diretamente relacionado com a producao de leite das maes (Peeters et al., 1992). A raca Lacaune tem sua origem atrelada a producao de animais de boa carcaca em diversas regioes da Franca e passou por um programa de melhoramento genetico que resultou em aumento da producao de leite e melhora da composicao da carcaca dos cordeiros destinados ao abate (Barillet et al., 2001). O presente trabalho teve o objetivo de avaliar a producao e a composicao do leite de tres genotipos de ovelhas Santa Ines, ½ Lacaune x ½ Santa Ines e ¾ Lacaune x ¼ Santa Ines ? e o desenvolvimento de seus cordeiros durante os quatro primeiros meses de lactacao.

Presença de Staphylococcus spp. produtores de enterotoxinas e da toxina da síndrome do choque tóxico em manipuladores de queijo de cabra

A total of 167 strains of Staphylococcus isolated from nasal cavities, oropharynx, palm of hands and subunguial of two goats cheese handlers were collected. The strains were pooled (45) according to the species similarity and place of origin and tested for the production of enterotoxins (SE) A, B, C, D and toxic shock toxin syndrome (TSST-1). It was observed that 62.2% of the pools presented the capacity to produce, individually or in association, SEA (33.3%); SEB (46.7%); SEC (8.9%); SED (4.4%) and TSST-1 (4.4%). From the enterotoxigenic pools, 96.4% corresponded to species negative coagulase (Staphylococcus epidermidis and Staphylococcus cohnii). The capacity to produce SE and TSST-1 by Staphylococcus spp. strains isolated from food handlers reaffirms its hole on the transmission of food poisoning, beyond emphasizing the indispensable necessity of the adoption of satisfactory hygienic and sanitary procedures during the food manufacture.