Assessment of the risk of raw milk consumption related to staphylococcal food poisoning

This study aimed to identify the risks of staphylococcal food poisoning due to the consumption of raw milk. Fifty-one farms in Londrina (PR) and 50 in Pelotas (RS) were analyzed, to determine the population of coagulase-positive staphylococci (UFC/ mL), as well as to verify the ability of producing Staphylococcal Enterotoxin A (SEA) by immunodifusion (OSP), the presence of the gene for the production of SEA (PCR) in the cultures, and the research of enterotoxin (SEA to SEE) in milk samples using ELISA commercial kit. Considering the 101 farms analyzed, 19 (18.8%) presented coagulase-positive staphylococci count above 105 UFC/mL. For the evaluation of the enterotoxigenic ability (SEA) by the OSP technique, six cultures coagulase-positive (5.5%) were positive to the test and identified as S. aureus. From the coagualse-negative sample, one (5.5%) was OSP positive. For the evaluation of the presence of the gene for EEA synthesis, 51 cultures of staphylococci were tested. From this total, 14 (27.45%) presented the gene, and from that, only 5 (9.81%) cultures were capable of expressing it in the technique of the OSP. The morphologic characteristic of the evaluated cultures that had enterotoxigenic capacity, from the 14 (33,3%) cultures that presented the gene for EEA production, 05 (11.9%) were characterized as typical cultures of S.aureus in Baird Parker agar. All the 12 milk samples studied for the presence of EEA to EEE in milk were negative. Thus, it can be concluded that there is extensive contamination of raw milk for staphylococci coagulase, however, most of the isolated strains were not enterotoxigenic or did not express such a characteristic. Only 9.81% of the tested colonies expressed the gene and effectively produced SEA. None of the samples had sufficient counts to produce detectable amounts of SEA. The milk samples did not present risk to cause staphylococcal food poisoning if consumed in natura until the collection moment.

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese

Aims To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. Methods and Results Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and a- and beta-haemolysis. Most isolates (93.0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93.0%) and efaA (83.7%). 53.5% of the isolates presented beta-haemolysis. Conclusions Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. Significance and Impact of the Study The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Revisiting a medical case of "stinging" in the human oral cavity caused by ingestion of raw squid (Cephalopoda: Teuthida): new data on the functioning of squid's spermatophores

Male squid produce intricate spermatophores that, when transferred to the female, undergo the spermatophoric reaction, a complex process of evagination that leads to the attachment of the spermatangium, that is, the everted spermatophore containing the sperm mass. While this process is still not completely understood, the medical literature includes several reports of "oral stinging" (i.e., punctured wounds in the human oral cavity) following consumption of raw male squid, which contains undischarged spermatophores able to inflict such wounds. Here, we revisit a recent medical report of oral stinging by Shiraki et al. (Pathol Int 61:749-751, 2011), providing an in-depth reanalysis of their histological biopsies and revealing vital information on the functioning of squid spermatophores. The morphology of the spermatangia attached within the oral cavity is similar to the condition found in spermatangia naturally attached to female squids. The spermatangia were able to superficially puncture the superficial layers of the oral stratified squamous epithelium, and numerous, minute stellate particles from the squid spermatophore were found adhered to the oral epithelium. These findings corroborate previous hypotheses on the functioning of squid spermatophores, namely that spermatophore attachment generally involves tissue scarification, and that stellate particles play a vital role in the attachment process. Moreover, spermatophore attachment is confirmed to be autonomous (i.e., performed by the spermatophore itself) in another squid species (possibly a loliginid), and the results strongly indicate that the attachment mechanism is not dependent upon a specialized epithelium, nor a mate's specific chemical stimulus. From the pathological point of view, the best prophylactic measure at present is the removal of the internal organs of the raw squid prior to its consumption.

Molecular detection of Streptococcus agalactiae in bovine raw milk samples obtained directly from bulk tanks

Mastitis is the most common infectious disease affecting dairy cattle; in addition, it remains the most economically important disease of dairy industries around the world. Streptococcus agalactiae, a contagious pathogen associated with subclinical mastitis, is highly infectious. This bacterium can cause an increase in bulk tank bacterial counts (BTBC) and bulk tank somatic cell counts (BTSCC). The microbiological identification of S. agalactiae in samples from bulk tanks is an auxiliary method to control contagious mastitis. Thus, there are some limitations for time-consuming cultures or identification methods and additional concerns about the conservation and transport of samples. Bulk tank samples from 247 dairy farms were cultured and compared through polymerase chain reaction (PCR), directed to 16S rRNA genes of S. agalactiae, followed by BTBC and S. agalactiae isolation. The mean value of BTBC was 1.08 x 10(6) CFU mL(-1) and the bacterium was identified through the microbiological method in 98 (39.7%; CI95% = 33.8-45.9%) and through PCR in 110 (44.5%; CI95% = 38.5-50.8%) samples. Results indicated sensitivity of 0.8571 +/- 0.0353 (CI95% = 0.7719-0.9196) and specificity of 0.8255 +/- 0.0311 (CI95% = 0.7549-0.8827). The lack of significant difference between microbiological and molecular results (kappa = 0.6686 +/- 0.0477 and CI95% = 0.5752-0.7620) indicated substantial agreement between the methods. This suggests that PCR can be used for bulk tank samples to detect contagious mastitis caused by S. agalactiae. (C) 2011 Elsevier Ltd. All rights reserved.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Characterization of polysaccharides from Hypnea spinella (Gigartinales) and Halopithys incurva (Ceramiales) and their effect on RAW 264.7 macrophage activity

[EN] Red algae have been reported to be an important source of polysaccharides with potential immunomodulatory properties. The objective of this study was to characterize the polysaccharides from Halopithys incurva and Hypnea spinella and to evaluate their effect on the synthesis of cytokines by murine cell line RAW 264.7 macrophages. Polysaccharides were obtained by N-cetylpyridiniumbromide precipitation and characterized by Fourier transform-infrared spectroscopy. Their effect on the activity of RAW 264.7 macrophages was examined by quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) production using enzyme-linked immunosorbent assays. The activation of the cytokine IL-6 and NO increased linearly as the concentration of polysaccharides from H. incurva and Hy. spinella increased. In general, the activation of IL-6 and NO was tenfold greater when macrophages were exposed to polysaccharides from H. incurva than when exposed to polysaccharides from Hy. spinella. In contrast, TNF-α concentration did not increase when macrophages were exposed to increasing polysaccharide levels. These results indicate that polysaccharides are strong cytokine IL-6 inducers.

Determination of bioactive and potentially toxic compounds in food: raw material analysis and shelf life evaluation

"Bioactive compounds" are extranutritional constituents that typically occur in small quantities in food. They are being intensively studied to evaluate their effects on health. Bioactive compounds include both water soluble compounds, such as phenolics, and lipidic substances such as n-3 fatty acids, tocopherols and sterols. Phenolic compounds, tocopherols and sterols are present in all plants and have been studied extensively in cereals, nuts and oil. n-3 fatty acids are present in fish and all around the vegetable kingdom. The aim of the present work was the determination of bioactive and potentially toxic compounds in cereal based foods and nuts. The first section of this study was focused on the determination of bioactive compounds in cereals. Because of that the different forms of phytosterols were investigated in hexaploid and tetraploid wheats. Hexaploid cultivars were the best source of esterified sterols (40.7% and 37.3% of total sterols for Triticum aestivum and Triticum spelta, respectively). Significant amounts of free sterols (65.5% and 60.7% of total sterols for Triticum durum and Triticum dicoccon, respectively) were found in the tetraploid cultivars. Then, free and bound phenolic compounds were identified in barley flours. HPLCESI/ MSD analysis in negative and positive ion mode established that barley free flavan-3- ols and proanthocyanidins were four dimers and four trimers having (epi)catechin and/or (epi)gallocatechin (C and/or GC) subunits. Hydroxycinnamic acids and their derivatives were the main bound phenols in barley flours. The results obtained demonstrated that barley flours were rich in phenolic compounds that showed high antioxidant activity. The study also examined the relationships between phenolic compounds and lipid oxidation of bakery. To this purpose, the investigated barley flours were used in the bakery production. The formulated oven products presented an interesting content of phenolic compounds, but they were not able to contain the lipid oxidation. Furthermore, the influence of conventional packaging on lipid oxidation of pasta was evaluated in n-3 enriched spaghetti and egg spaghetti. The results proved that conventional packaging was not appropriated to preserve pasta from lipid oxidation; in fact, pasta that was exposed to light showed a high content of potentially toxic compounds derived from lipid oxidation (such as peroxide, oxidized fatty acids and COPs). In the second section, the content of sterols, phenolic compounds, n-3 fatty acids and tocopherols in walnuts were reported. Rapid analytical techniques were used to analyze the lipid fraction and to characterize phenolic compounds in walnuts. Total lipid chromatogram was used for the simultaneous determination of the profile of sterols and tocopherols. Linoleic and linolenic acids were the most representative n-6 and n-3 essential dietary fatty acids present in these nuts. Walnuts contained substantial amounts of γ- and δ-tocopherol, which explained their antioxidant properties. Sitosterol, Δ5-avenasterol and campesterol were the major free sterols found. Capillary electrophoresis coupled to DAD and microTOF was utilized to determine phenolic content of walnut. A new compound in walnut ((2E,4E)- 8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester, [M−H]− 403.161m/z) with a structure similar to glansreginins was also identified. Phenolic compounds corresponded to 14–28% of total polar compounds quantified. Aglycone and glycosylated ellagic acid represented the principal components and account for 64–75% of total phenols in walnuts. However, the sum of glansreginins A, B and ((2E,4E)-8-hydroxy- 2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester was in the range of 72–86% of total quantified compounds.

Vegetables biofortified with iodine: chemical, agronomic and qualitative aspects of raw and fried potatoes

Iodine is an essential microelement for human health because it is a constituent of the thyroid hormones that regulate growth and development of the organism. Iodine Deficiency Disorders (IDDs) are believed to be one of the commonest preventable human health problems in the world today, according to the World Health Organization: that diseases include endemic goiter, cretinism and fetal abnormalities, among others, and they are caused by lack of iodine in the diet, that is the main source of iodine. Since iodine intake from food is not enough respect to human needs, this can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentration and/or bioavailability of mineral elements in the edible portions of crops through agricultural intervention or genetic selection (biofortification). The introduction of iodized salt is a strategy widely used and accepted to eradicate iodine deficiency, because it is an inexpensive source of stable iodine. Since the intake of salt, though iodized, must still be limited according to the risk of cardiovascular disease, so the increase of iodine content in plants for the production of functional foods is representing a field of study of particular interest and a potential market. In Italy potatoes enriched with iodine are produced by a patented procedure of agronomic biofortification for the fresh market since several years, furthermore they are recently accepted and recommended by Italian Thyroid Association, as an alternative source of iodine. Researches performed during the PhD course intended to characterize this innovative vegetables products, focusing the attention on different aspects, such as chemistry, agriculture, and quality of fresh and fried potatoes. For this purpose, lipid fraction of raw material was firstly investigated, in order to assess whether the presence of iodine in plant metabolism can affect fatty acid or sterol biosynthesis, according to the hypothesis that iodine can be bounded to polyunsaturated fatty acids of cell membranes, protecting them from peroxydation; phytosterols of plant sterol are also studied because their importance in reducing serum cholesterol, especially in potato plant sterols are also involved in synthesis of glycoalkaloid, a family of steroidal toxic secondary metabolites present in plants of the Solanaceae family. To achieve this goal chromatographic analytical techniques were employed to identify and quantify fatty acids and sterols profile of common and iodine enriched row potatoes. Another aim of the project was to evaluate the effects of frying on the quality of iodine-enriched and common potatoes. Since iodine-enriched potatoes are nowadays produced only for the fresh market, preliminary trials of cultivation under controlled environment were carried out to verify if potato varieties suitable for processing were able to absorb and accumulate iodine in the tuber. In a successive phase, these varieties were grown in the field, to evaluate their potential productivity and quality at harvest and after storage. The best potato variety to be destined for processing purposes, was finally subjected to repeated frying cycles; the effects of lipid oxidation on the composition and quality of both potatoes and frying oil bath were evaluated by chromatographic and spectrophotometric analytical techniques. Special attention were paid on volatile compounds of fried potatoes.

By-products of the dairy farming as raw material for the biotechnology production of polyhydroxyalkanoates

I Poliidrossialcanoati (PHA) sono poliesteri completamente biodegradabili, prodotti da microrganismi come fonte di energia e di carbonio per la sintesi di nuovo materiale cellulare, utilizzando come substrato materie prime rinnovabili. Questi poliesteri sono considerati potenziali candidati per la sostituzione delle materie plastiche convenzionali. Tuttavia, i più alti costi di produzione dei PHA in confronto a quelli delle materie plastiche derivanti dal petrolio, rappresentano il principale ostacolo per la parziale sostituzione di questi ultimi con i biopolimeri. Gli alti costi sono principalmente dovuti all'utilizzo di colture microbiche pure (in cui sia presente un solo ceppo batterico) e substrati puri e costosi. Nell'ultimo decennio è stato sviluppato un processo di produzione a tre stadi alternativo e potenzialmente a minor costo, basato sull'utilizzo di colture microbiche miste (Mixed Microbials Culture, MMC) e una varietà di substrati organici a costo contenuto o nullo, quali alcuni rifiuti dell’industria agro-alimentare. Il presente studio si è concentrato sulla prima fase del processo di produzione dei PHA da colture miste, la fermentazione acidogenica, utilizzando siero di latte come fonte di carbonio per produrre acidi organici. In particolare questo lavoro ha avuto come obiettivo quello di studiare come diverse condizioni operative utilizzate nella fase di fermentazione acidogenica possono influenzare la concentrazione e il profilo degli acidi organici prodotti. Sono stati valutati anche gli effetti dei diversi profili degli acidi organici sulla fase di selezione della coltura microbica, in termini di capacità di stoccaggio di PHA e composizione polimerica.

Development of an innovative pyrolysis plant for the production of secondary raw materials

This project was born with the aim of developing an environmentally and financially sustainable process to dispose of end-life tires. In this perspective was devised an innovative static bed batch pilot reactor where pyrolysis can be carried out on the whole tires in order to recover energy and materials and simultaneously save the energy costs of their shredding. The innovative plant is also able to guarantee a high safety of the process thanks to the presence of a hydraulic guard. The pilot plant was used to pyrolyze new and end-life tires at temperatures from 400 to 600°C with step of 50°C in presence of steam. The main objective of this research was to evaluate the influence of the maximum process temperature on yields and chemical-physics properties of pyrolysis products. In addition, in view of a scale-up of the plant in continuous mode, the influence of the nature of several different tires as well as the effects of the aging on the final products were studied. The same pilot plant was also used to carry out pyrolysis on polymeric matrix composites in order to obtain chemical feedstocks from the resin degradation together with the recovery of the reinforcement in the form of fibers. Carbon fibers reinforced composites ad fiberglass was treated in the 450-600°C range and the products was fully characterized. A second oxidative step was performed on the pyrolysis solid residue in order to obtain the fibers in a suitable condition for a subsequent re-impregnation in order to close the composite Life Cycle in a cradle-to-cradle approach. These investigations have demonstrated that steel wires, char, carbon and glass fibers recovered in the prototypal plant as solid residues can be a viable alternative to pristine materials, making use of them to obtain new products with a commercial added value.

The Appellate Body Approach to the Applicability of Article XX GATT In the Light of China - Raw Materials: A Missed Opportunity?

This article attempts to analyse and investigate the implications of the approach to the applicability of Article XX GATT adopted in the recent China – Raw Materials. Using the decision on the non-availability of Article XX defences for violations of China’s WTO-plus commitments on export duties as a backdrop, it scrutinizes the more general, ‘systemic’ approach to the applicability of Article XX exceptions developed by theWTO dispute settlement bodies, and sheds light on the implications of such approach with respect to the relationship between GATT 1994 andWTO obligations arising from different instruments of theWTOAgreement, such as new members’ accession protocols. It also suggests that an exception to this general approach could be envisaged when the fundamental environmental goals protected under Article XX b) and g) are at stake.