Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.     

Arsenite oxidation by a poorly crystalline manganese-oxide. 2. Results from X-ray absorption spectroscopy and X-ray diffraction

Arsenite (As(III)) oxidation by manganese oxides (Mn-oxides) serves to detoxify and, under many conditions, immobilize arsenic (As) by forming arsenate (As(V)). As(III) oxidation by Mn(IV)-oxides can be quite complex, involving many simultaneous forward reactions and subsequent back reactions. During As(III) oxidation by Mn-oxides, a reduction in oxidation rate is often observed, which is attributed to Mn-oxide surface passivation. X-ray absorption spectroscopy (XAS) and X-ray diffraction (XRD) data show that Mn(II) sorption on a poorly crystalline hexagonal birnessite (δ-MnO?) is important in passivation early during reaction with As(III). Also, it appears that Mn(III) in the δ-MnO? structure is formed by conproportionation of sorbed Mn(II) and Mn(IV) in the mineral structure. The content of Mn(III) within the δ-MnO? structure appears to increase as the reaction proceeds. Binding of As(V) to δ-MnO? also changes as Mn(III) becomes more prominent in the δ-MnO ? structure. The data presented indicate that As(III) oxidation and As(V) sorption by poorly crystalline δ-MnO? is greatly affected by Mn oxidation state in the δ-MnO? structure.     

Degradation of lead-contaminated lignocellulosic waste by Phanerochaete chrysosporium and the reduction of lead toxicity

Lead, as one of the most hazardous heavy metals to the environment interferes with lignocellulosic biomass bioconversion and carbon cycles in nature. The degradation of lead-polluted lignocellulosic waste and the restrain of lead hazards by solid-state fermentation with Phanerochaete chrysosporium were studied. Phanerochaete chrysosporium effectively degraded lignocellulose, formed humus and reduced active lead ions, even at the concentration of 400 mg/kg dry mass of lead. The highest lignocellulose degradation (56.8%) and organic matter loss (64.0%) were found at the concentration of 30 mg/kg of lead, and at low concentration of lead the capability of selective lignin biodegradation was enhanced. Microbial growth was delayed in polluted substrate at the initial stage of fermentation, and organic matter loss is correlated positively with microbial biomass after 12 day fermentation. It might be because Phanerochaete chrysosporium developed active defense mechanism to alleviate the lead toxicity. Scanning electron micrographs with energy spectra showed that lead was immobilized via two possible routes: adsorption and cation exchange on hypha, and the chelation by fungal metabolite. The present findings will improve the understandings about the degradation process and the lead immobilization pathway, which could be used as references for developing a fungi-based treatment technology for metal-contaminated lignocellulosic waste.     

Disentangling oil weathering at a marine seep using GC x GC: broad metabolic specificity accompanies subsurface petroleum biodegradation

Natural seeps contribute nearly half of the oil entering the coastal ocean. However, environmental fate studies generally monitor fewer than 5% of these petroleum compounds. Hence, the rates and relevance of physical, chemical, and biological weathering processes are unknown for the large majority of hydrocarbons, both released from natural seeps and also from human activities. To investigate the specific compositional changes occurring in petroleum during subsurface degradation and submarine seepage, we studied the natural oil seeps offshore Santa Barbara, California with comprehensive, two-dimensional gas chromatography (GC x GC). With this technique, we quantified changes in the molecular diversity and abundance of hydrocarbons between subsurface reservoirs, a proximal sea floor seep, and the sea surface overlying the seep. We also developed methods to apportion hydrocarbon mass losses due to biodegradation, dissolution, and evaporation, for hundreds of tracked compounds that ascended from the subsurface to the sea floor to the sea surface. The results provide the first quantitative evidence of broad metabolic specificity for anaerobic hydrocarbon degradation in the subsurface and reveal new trends of rapid hydrocarbon evaporation at the sea surface. This study establishes GC x GC as a powerful technique for differentiating biological and physical weathering processes of complex mixtures at a molecular level.     

Post-slaughter traceability

Traceability programs can cover the whole of life, or parts of it, for individual animals or groups/lots of animals. Of 13 country or community traceability programs for cattle/beef, 11 are mandatory (4 encompass, or are scheduled to encompass, birth to retail; 7 cover birth to slaughter) while 2 are voluntary and encompass birth to slaughter. Of 10 country or community traceability programs for swine/pork, 2 are mandatory (1 covers birth to retail; 1 covers birth to slaughter) while 8 are voluntary. Of 6 country or community traceability programs for sheep/sheep-meat, 3 are mandatory (1 encompasses birth to retail; 2 encompass birth to slaughter) while 3 are voluntary. Mandatory birth to retail programs that include "post-slaughter individual animal identification (IAID) traceability" have been implemented for cattle/beef, swine/pork and sheep/sheep-meat by the European Union and for cattle/beef by Japan. Many of the voluntary as well as mandatory, birth to slaughter traceability programs for all three species are presumed (though that is not specified) to include "post-slaughter group/lot identification (GLID) traceability" - e.g., those qualifying products for shipment to the European Union. "Post-slaughter IAID traceability" can be accomplished in very-small, small, medium, large and very-large packing plants using single-carcass processing units, tagging and separation/segregation, and/or deoxyribonucleic acid (DNA) fingerprinting technology but all of these approaches are time-consuming and costly; and, to-date, in most countries, there has been no reason compelling enough to cause industry to adopt such protocols or technology.     

Inhibition of protease activity. Part 1. The effect on tenderness and indicators of proteolysis in ovine muscle

To examine the effect of particular enzyme groups on tenderness specific cysteine protease inhibitors were injected into muscle early post-mortem. The protease enzyme inhibitor E-64 was injected into the m. longissimus thoracis et lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). To create variation in the rate of pH decline alternate carcasses were electrically stimulated (low voltage). The LTL was divided into cranial and caudal portions and aged for 1 or 2 days. Muscle samples at 1 day post-mortem were used for measurement of osmolality and sarcomere length (n=48), and others at 1 and 2 days post-mortem for shear force determination (n=96). The myofibrillar fragmentation index (MFI) was determined on samples taken at pH 6.2 and 1 and 2 days post-mortem (n=144). Other muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). These samples were used for determination of protein solubility and the concentration of free amino acids. Stimulation caused a faster (P<0.05) decline in pH. There was no effect of stimulation (P>0.05) on shear force values, but injection of inhibitor and ageing both had effects (P<0.001). The inhibitor E-64 prevented any improvement in tenderness with ageing, whereas the inhibitor Z-Phe-Ala-CHN(2) and the control samples showed a similar ageing response. In the latter two treatments there was an average reduction of 1 kg in shear between 1 and 2 days post-mortem, whilst the inhibitor E-64 maintained shear force on average 2 kg higher than control samples. Injection and ageing had an effect on MFI (P<0.001) and there was an interaction (P<0.05) between stimulation and ageing for MFI, such that as stimulated muscle aged the rate of change of MFI was greater. There was an interaction between injection and ageing (P<0.05) for protein solubility such that samples treated with E-64 showed a minimal increase in protein solubility with ageing, whereas in samples treated with Z-Phe-Ala-CHN(2) and the control samples there was a significant increase. There was also an interaction between stimulation and ageing such that between sampling at pH 6.0 and 2 days post-mortem, stimulated muscle exhibited greater solubility (P<0.05). There were no effects (P>0.05) on the concentration of free amino acids. The evidence indicated that the cysteine proteases were responsible for post-mortem proteolysis and tenderisation, in particular the calpains, whereas the cathepsins (B and L) were unlikely to contribute to proteolysis and subsequent tenderisation in meat.     

Reactivity of Pb(II) at the Mn(III,IV) (oxyhydr)oxide--water interface

In this study, the reactivity of lead (Pb(II)) on naturally occurring Mn(III,IV) (oxyhydr)oxide minerals was evaluated using kinetic, thermodynamic, and spectroscopic investigations. Aqueous Pb(II) was more strongly adsorbed to birnessite (delta-MnO1.7) than to manganite (gamma-MnOOH) under all experimental conditions. The isoteric heat of Pb adsorption (delta HT) or birnessite was 94 kJ mol-1 at a surface loading of 1.1 mmol g-1, and decreased with increasing adsorption density. This indicated that adsorption was an endothermic process and that birnessite possessed heterogeneous sites of reactivity for Pb. X-ray absorption fine structure (XAFS) spectra revealed that Pb was adsorbed as inner-sphere complexes on both birnessite and manganite with no evidence to suggest oxidation as an operative sorption mechanism. Lead appeared to coordinate to vacancy sites in the birnessite layer structure with concurrent release of Mn to solution, which resulted in a greater number of second shell Mn scatterers in Pb-birnessite when compared to Pb-manganite samples. The difference in Pb coordination apparently explained the contrasting desorption behavior between the two Mn minerals. These results have significant implications for Pb partitioning in soil environments containing solid-phase Mn(III,IV) (oxyhydr)oxides.     

The effects of alkaline hydrogen peroxide treatment on the nutritional value of tripe

Samples of tripe obtained after commercial processing with alkaline hydrogen peroxide were compared with raw tripe for nutritional quality. A large number (108) of commercial tripe samples were found to vary markedly in their protein content from 8·4 to 19·3%. The moisture content was negatively correlated with the pH of the tripe samples. However, the amino acid analyses and rat bioassays indicated that the nutritional value of the protein was not significantly decreased by the processing. Oxidation of sulphur amino acids and formation of lysino-alanine were not evident. Thiamine was completely destroyed by the processing.