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Physiological probes directly bind to particular molecules in the cell or are markers of metabolic activities (such as enzyme activity and membrane poten- tial). These probes are non-specific, i.e. their action is independent of the type and the identity of the cell. Depending on whether a stain is membrane permeant or impermeant it will label both viable and dead cells or dead cells only, respec- tively. ChemChrome V6 (formerly ChemChrome B or V3) contains carboxyfluo- rescein diacetate (Fig. 4), a nonfluorescent compound that is taken up by metabolically active cells and cleaved by intracellular esterases to yield an intensely green fluorescent product (carboxyfluorescein). When used in conjunc- tion with an appropriate labelling buffer (ChemSol B12), the fluorescent car- boxyfluorescein is only retained in cells with an intact cytoplasmatic membrane (Catala et al. 1999; Parthuisot et al. 2000). As the dye will rapidly leak from dead cells because of their damaged membranes, ChemChrome V6 functions as an activity and cell integrity probe that measures both enzymatic activity and cell- membrane integrity.

Figure 4 Physiological probes directly bind to particular molecules in the cell or are markers of metabolic activities (such as enzyme activity and membrane poten- tial). These probes are non-specific, i.e. their action is independent of the type and the identity of the cell. Depending on whether a stain is membrane permeant or impermeant it will label both viable and dead cells or dead cells only, respec- tively. ChemChrome V6 (formerly ChemChrome B or V3) contains carboxyfluo- rescein diacetate (Fig. 4), a nonfluorescent compound that is taken up by metabolically active cells and cleaved by intracellular esterases to yield an intensely green fluorescent product (carboxyfluorescein). When used in conjunc- tion with an appropriate labelling buffer (ChemSol B12), the fluorescent car- boxyfluorescein is only retained in cells with an intact cytoplasmatic membrane (Catala et al. 1999; Parthuisot et al. 2000). As the dye will rapidly leak from dead cells because of their damaged membranes, ChemChrome V6 functions as an activity and cell integrity probe that measures both enzymatic activity and cell- membrane integrity.

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Fig. 1 Schematic overview of solid-phase cytometry. Steps include: membrane filtration, fluores- cent labelling, scanning, data analysis by a computer and microscopic validation Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry
Fig. 1 Schematic overview of solid-phase cytometry. Steps include: membrane filtration, fluores- cent labelling, scanning, data analysis by a computer and microscopic validation Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry
Fig. 2 Overview of the software parameters used by the computer to discriminate among micro- organisms and autofluorescent particles. The four main discriminants are size, specific intensity, color ratio and signal pattern of the detected fluorescent spot
Fig. 2 Overview of the software parameters used by the computer to discriminate among micro- organisms and autofluorescent particles. The four main discriminants are size, specific intensity, color ratio and signal pattern of the detected fluorescent spot
Fig. 4 Cleavage of ChemChrome V6 by esterases to yield the green fluorescent carboxyfluo- racrain 8-glucuronidase activity resulting from the use of a proprietary cocktail of inducers and stabilizers.
Fig. 4 Cleavage of ChemChrome V6 by esterases to yield the green fluorescent carboxyfluo- racrain 8-glucuronidase activity resulting from the use of a proprietary cocktail of inducers and stabilizers.
Fig. 5 Schematic presentation of tyramide signal amplification (TSA) Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry
Fig. 5 Schematic presentation of tyramide signal amplification (TSA) Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry
Table 1 (continued)
Table 1 (continued)
Fig. 1 Hydrostratigraphic section through wells NN 1, 4, 7, 8, 10, 12. (1) Loam with gravel, pebble, basalt detritus, waterless. (2) Basalt detritus, pebble filled with loam. (3) Monolithe basalt. (4) Fractured basalt Vulnerability Assessment and Mapping of Akounq Groundwater Body, Armenia
Fig. 1 Hydrostratigraphic section through wells NN 1, 4, 7, 8, 10, 12. (1) Loam with gravel, pebble, basalt detritus, waterless. (2) Basalt detritus, pebble filled with loam. (3) Monolithe basalt. (4) Fractured basalt Vulnerability Assessment and Mapping of Akounq Groundwater Body, Armenia
Table 1 Numerical scores assigned to water level below land surface (b.1.s.) and to soils according their thickness and lithology
Table 1 Numerical scores assigned to water level below land surface (b.1.s.) and to soils according their thickness and lithology
Table 2 Classification of water-table aquifer by vulnerability degree
Table 2 Classification of water-table aquifer by vulnerability degree
Table 3 Evaluation of water-table aquifer vulnerability by bacteriological contamination Period of vital functions of bacteria under slow contamination is taken 200 days, under mass infection (natural or man-caused disaster) or continuous contaminant spill conditions it is taken 400 days. Six categories of ground water vulnerability to bacteriological contamination have been distinguished (Table 3): Quantitative assessment of confined aquifer vulnerability that is first from land surface is carried on the base of two parameters: total thickness of overlain soils that have slow infiltration rates (m,) and by levels of water table (H.) and estab-
Table 3 Evaluation of water-table aquifer vulnerability by bacteriological contamination Period of vital functions of bacteria under slow contamination is taken 200 days, under mass infection (natural or man-caused disaster) or continuous contaminant spill conditions it is taken 400 days. Six categories of ground water vulnerability to bacteriological contamination have been distinguished (Table 3): Quantitative assessment of confined aquifer vulnerability that is first from land surface is carried on the base of two parameters: total thickness of overlain soils that have slow infiltration rates (m,) and by levels of water table (H.) and estab-
Table 4 Water-table aquifer intrinsic vulnerability assessment by numerical scores
Table 4 Water-table aquifer intrinsic vulnerability assessment by numerical scores
Table 5 Water-table aquifer natural vulnerability assessment from bacteriological contamination
Table 5 Water-table aquifer natural vulnerability assessment from bacteriological contamination
Table 6 Confined aquifer natural vulnerability assessment
Table 6 Confined aquifer natural vulnerability assessment
performed in order to increase the cell numbers of the pathogen to a detectable level and to allow recovery of injured cel three parameters: (i) the temperature, (ii) s. The efficiency of this step rely on the length of incubation and (iii) the the broth composition. While growth of STEC is usually carried out at 37°C, some authors also incubate the broth at 4 °C (Vimont et al. 2006). The length of incubation is also variable, and may vary depending on the protocol and the matrice enriched, ranging from 6 to 24 h. Possé et al. (2008a) performed an enrichment method including a pre-enric hment phase at 42°C and an enrich- ment at 37°C. The two most employed enrichment media for E. coli O157:H7 and other STEC serogroups are modified trypton soy broth (mTSB) and E. coli broth (EC). These media may be supplemented with various selective agents such as bile salts, potassium tellurite and is the most commonly used (Vimont et al. antibiotics among which novobiocin 2006). However, the use of antibiot- ics may interfere with STEC growth and concentration of these additive has to be carefully chosen (Foster et al. 2003; Vimont et al. 2006). The International Standard Organization (ISO) reference method for isolation of
performed in order to increase the cell numbers of the pathogen to a detectable level and to allow recovery of injured cel three parameters: (i) the temperature, (ii) s. The efficiency of this step rely on the length of incubation and (iii) the the broth composition. While growth of STEC is usually carried out at 37°C, some authors also incubate the broth at 4 °C (Vimont et al. 2006). The length of incubation is also variable, and may vary depending on the protocol and the matrice enriched, ranging from 6 to 24 h. Possé et al. (2008a) performed an enrichment method including a pre-enric hment phase at 42°C and an enrich- ment at 37°C. The two most employed enrichment media for E. coli O157:H7 and other STEC serogroups are modified trypton soy broth (mTSB) and E. coli broth (EC). These media may be supplemented with various selective agents such as bile salts, potassium tellurite and is the most commonly used (Vimont et al. antibiotics among which novobiocin 2006). However, the use of antibiot- ics may interfere with STEC growth and concentration of these additive has to be carefully chosen (Foster et al. 2003; Vimont et al. 2006). The International Standard Organization (ISO) reference method for isolation of
ther sorbitol-non-fermenting bacterial species tested. This agar was tested on attle rectal swabs and has increased the rate of STEC O157 isolation. Cefixime ellurite Sorbitol MacConkey Agar (CT-SMAC) is now the media of choice in the SO standard protocol (ISO 16654) for E. coli O157 although this medium has been eported to fail to isolate two sorbitol-fermenting STEC O157:H- on HUS patients Karch et al. 1996).
ther sorbitol-non-fermenting bacterial species tested. This agar was tested on attle rectal swabs and has increased the rate of STEC O157 isolation. Cefixime ellurite Sorbitol MacConkey Agar (CT-SMAC) is now the media of choice in the SO standard protocol (ISO 16654) for E. coli O157 although this medium has been eported to fail to isolate two sorbitol-fermenting STEC O157:H- on HUS patients Karch et al. 1996).
Table 3 Commercialized immunological methods for the detection and isolation of E. coli O157:H7 in food. (Vernozy-Rozand et al., 2002)
Table 3 Commercialized immunological methods for the detection and isolation of E. coli O157:H7 in food. (Vernozy-Rozand et al., 2002)
indicator for viability of E. coli O157:H7 cells under typical growth conditions before and after thermal treatment. They found that 7fbE, stx/, hly and 16S RNA amplicons were detected during all growth phase. But only the RT-PCR signal of 16S rRNA and /fbE was stable. After heating at 60°C for 20 min, all the mRNA species were degraded, except the rRNA. The authors suggested that the 7fbE gene is the most appropriate target for the detection of viable E. coli O157:H7. However,
indicator for viability of E. coli O157:H7 cells under typical growth conditions before and after thermal treatment. They found that 7fbE, stx/, hly and 16S RNA amplicons were detected during all growth phase. But only the RT-PCR signal of 16S rRNA and /fbE was stable. After heating at 60°C for 20 min, all the mRNA species were degraded, except the rRNA. The authors suggested that the 7fbE gene is the most appropriate target for the detection of viable E. coli O157:H7. However,
Table 4 (continued)
Table 4 (continued)
Fig. 2 AFSSA screening strategy to detect the five major STEC serogroup involved in human cases (EFSA 2007)
Fig. 2 AFSSA screening strategy to detect the five major STEC serogroup involved in human cases (EFSA 2007)
Table 1 Occurrence of enterococci in the gastro-intestinal tract ++: usual; + : frequent; (+): occasional; — : not mentioned (According to Klein (2003))
Table 1 Occurrence of enterococci in the gastro-intestinal tract ++: usual; + : frequent; (+): occasional; — : not mentioned (According to Klein (2003))
++: usual; + : frequent; (+): occasional; — : not mentioned; ND: not investigated (Adapted from Klein 2003) Table 2. Enterococci in food
++: usual; + : frequent; (+): occasional; — : not mentioned; ND: not investigated (Adapted from Klein 2003) Table 2. Enterococci in food
Table 3. European cheese products which involved enterococci
Table 3. European cheese products which involved enterococci
Table 3 (continued) The enterococci contribute to the ripening and aroma development of cheese due to their proteolytic and esterolytic activities, as well as their diacetyl production by citrate metabolism (Centeno et al. 1999). The presence and growth of enterococci in cheeses and sausages results in organoleptically unique products, which contrib- ute to the local cuisine and the region’s heritage. Due to interlinked European and worldwide markets, these cheeses are widely distributed and are internationally considered as delicacies (Franz et al. 1999). ee q49,% 7 is 7 - x q aie 7 ‘es: n
Table 3 (continued) The enterococci contribute to the ripening and aroma development of cheese due to their proteolytic and esterolytic activities, as well as their diacetyl production by citrate metabolism (Centeno et al. 1999). The presence and growth of enterococci in cheeses and sausages results in organoleptically unique products, which contrib- ute to the local cuisine and the region’s heritage. Due to interlinked European and worldwide markets, these cheeses are widely distributed and are internationally considered as delicacies (Franz et al. 1999). ee q49,% 7 is 7 - x q aie 7 ‘es: n
Fig. 1 Dendrogram based on cluster analysis of digitized (GTG).-PCR fingerprints of type, refer- ence and Bryndza cheese strains. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correlation coefficient. Numbered clusters (1-9) correspond to identified species of Enterococcus sp. and Lactococcus sp.
Fig. 1 Dendrogram based on cluster analysis of digitized (GTG).-PCR fingerprints of type, refer- ence and Bryndza cheese strains. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correlation coefficient. Numbered clusters (1-9) correspond to identified species of Enterococcus sp. and Lactococcus sp.
Fig. 4 (continued) (b) PFGE, (GTG)5-PCR, ERIC-PCR fingerprints of 82 E. faecium isolates. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correla- tion coefficient (For further legends see Fig. 4a)
Fig. 4 (continued) (b) PFGE, (GTG)5-PCR, ERIC-PCR fingerprints of 82 E. faecium isolates. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correla- tion coefficient (For further legends see Fig. 4a)
Fig. 5 Antibiotic resistance behaviour of 308 enterococci isolated from Bryndza. Ampicillin (AMP), streptomycin (STR), gentamicin (GEN), vancomycin (VAN), teicoplanin (TEI), rifampi- cin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)
Fig. 5 Antibiotic resistance behaviour of 308 enterococci isolated from Bryndza. Ampicillin (AMP), streptomycin (STR), gentamicin (GEN), vancomycin (VAN), teicoplanin (TEI), rifampi- cin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)
Fig. 6 Incidence of sensitive, intermediary and resistant E. faecium strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT) Figures 6-8 illustrate antibiotic resistance patterns of E. faecium, E. faecali: ind E. durans isolates from Bryndza cheese. No one of the E. faecium, E. duran: ind E. faecalis iso ates were resistant to ampicillin, streptomycin, gentamicin, van -omycin, and teicoplanin. Thirty six percent of the E. faecium isolates and 22% o he E. faecalis iso ‘esistance to rifam ates were resistant to erythromycin. E. faecium showed simila picin (31%) as E. faecalis (29%). Both E. faecium and E. faecali strains exhibited the same resistance to ciprofloxacin (2%). E. durans isolate showed very low level of resistance to rifampicin, erythromycin, ciprofloxacin, an 1itrofurantoin (1-4 I%). E. faecium, E. faecalis and E. durans were also found to b he predominant s pecies recovered from naturally ripened European cheeses an
Fig. 6 Incidence of sensitive, intermediary and resistant E. faecium strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT) Figures 6-8 illustrate antibiotic resistance patterns of E. faecium, E. faecali: ind E. durans isolates from Bryndza cheese. No one of the E. faecium, E. duran: ind E. faecalis iso ates were resistant to ampicillin, streptomycin, gentamicin, van -omycin, and teicoplanin. Thirty six percent of the E. faecium isolates and 22% o he E. faecalis iso ‘esistance to rifam ates were resistant to erythromycin. E. faecium showed simila picin (31%) as E. faecalis (29%). Both E. faecium and E. faecali strains exhibited the same resistance to ciprofloxacin (2%). E. durans isolate showed very low level of resistance to rifampicin, erythromycin, ciprofloxacin, an 1itrofurantoin (1-4 I%). E. faecium, E. faecalis and E. durans were also found to b he predominant s pecies recovered from naturally ripened European cheeses an
Fig. 7 Incidence of sensitive, intermediary and resistant E. faecalis strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT)
Fig. 7 Incidence of sensitive, intermediary and resistant E. faecalis strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT)
Fig. 8 Incidence of sensitive, intermediary and resistant E. durans strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)
Fig. 8 Incidence of sensitive, intermediary and resistant E. durans strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)
Mili, LUALLY OLIULEE La IVIL ALI 1UUU SESE A eye: S. aureus is a bacterium which has gained notoriety over recent years due tc its ability to evolve new virulent and drug resistant strains (Chambers 2001) Strains of this species carry in their genomes genes encoding virulence factors suck as enterotoxins, toxic shock syndrome toxin, exfoliative toxins and immune evasior factors. These genes are often carried on mobile genetic elements including bacte- riophages. In recent years, increasingly virulent strains of S. aureus have emergec in the community, causing severe skin and soft tissue infections and a lethal forn of hemolytic pneumonia in children (Gillet et al. 2002). These new strains are asso: ciated with a bi-component toxin called Panton-Valentine leukocidin (PVL). The increasing levels of antibiotic resistance and the emergence of epidemic strains 0’ bacterial pathogens over the last decade highlight the adaptability of bacteria anc the remarkable speed of bacterial evolution (Enright et al. 2002).
Mili, LUALLY OLIULEE La IVIL ALI 1UUU SESE A eye: S. aureus is a bacterium which has gained notoriety over recent years due tc its ability to evolve new virulent and drug resistant strains (Chambers 2001) Strains of this species carry in their genomes genes encoding virulence factors suck as enterotoxins, toxic shock syndrome toxin, exfoliative toxins and immune evasior factors. These genes are often carried on mobile genetic elements including bacte- riophages. In recent years, increasingly virulent strains of S. aureus have emergec in the community, causing severe skin and soft tissue infections and a lethal forn of hemolytic pneumonia in children (Gillet et al. 2002). These new strains are asso: ciated with a bi-component toxin called Panton-Valentine leukocidin (PVL). The increasing levels of antibiotic resistance and the emergence of epidemic strains 0’ bacterial pathogens over the last decade highlight the adaptability of bacteria anc the remarkable speed of bacterial evolution (Enright et al. 2002).
Table 3. Comparison of genotyping systems applied to Staphylococcus aureus (modified according to Witte et al. 2006) The main typing systems used for epidemiological purposes in S. aureus are summarized in Table 3. The methods are compared in respect to their reproduc- bility and discriminatory power. Typing by means of PCR methods is rapid and nexpensive and provides results which are sufficient for quick analysis concerning he relatedness of nosocomial isolates. Spa sequence typing has the advantage of 1igh reproducibility; the sequence data are portable and can be stored in databases. Vloreover, spa typing has a good discriminatory power and can, therefore, replace ymal macrorestriction analysis.
Table 3. Comparison of genotyping systems applied to Staphylococcus aureus (modified according to Witte et al. 2006) The main typing systems used for epidemiological purposes in S. aureus are summarized in Table 3. The methods are compared in respect to their reproduc- bility and discriminatory power. Typing by means of PCR methods is rapid and nexpensive and provides results which are sufficient for quick analysis concerning he relatedness of nosocomial isolates. Spa sequence typing has the advantage of 1igh reproducibility; the sequence data are portable and can be stored in databases. Vloreover, spa typing has a good discriminatory power and can, therefore, replace ymal macrorestriction analysis.
—: 290% strains negative; +: 290% strains positive Table 1 Bacillus anthracis, B. cereus, B. thuringiensis characteristics
—: 290% strains negative; +: 290% strains positive Table 1 Bacillus anthracis, B. cereus, B. thuringiensis characteristics
Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods associated to food poisoning only in recent years (van Netten et al. 1990). B. cereus, B. thuringiensis and B. anthracis are important species for human healt as they carry genes encoding virulence factors affecting pathogenesis (Sergeev et al. 2006). As some B. thuringiensis strains have been reported to possess genes known to be involved in B. cereus pathogenesis (Hansen and Hendriksen 2001) a differentiation of B. cereus ad B. thuringiensis detected in food products could be a real contri- bution to the collection of epidemiological data. B. cereus, B. thuringiensis and B. anthracis have been subjected to several genomic studies to understand the taxo- nomic relationships. Data reported by Helgason et al. (2000a) reveal that B. cereus, B. thuringiensis and B. anthracis are indistinguishable using MEE or sequence analyses of nine genes, according with data obtained using DNA-DNA hybridization analysis by Kaneko et al. (1978) and Priest et al. (1994). Genetic evidence indicates that B. cereus, B. thuringiensis and B. anthracis are one s Cherif et al. (2003a) grouped B. anthracis in a separa pecies (Helgason et al. 2000b). te branch from B. cereus and B. thuringiensis on the basis of the phylogenetic relationship between the species of the B. cereus group using the long ITS containing t DNA. Data on ITS sequences confirm the difficulty in distinguishing B. cereus and B. thuringiensis apart from
Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods associated to food poisoning only in recent years (van Netten et al. 1990). B. cereus, B. thuringiensis and B. anthracis are important species for human healt as they carry genes encoding virulence factors affecting pathogenesis (Sergeev et al. 2006). As some B. thuringiensis strains have been reported to possess genes known to be involved in B. cereus pathogenesis (Hansen and Hendriksen 2001) a differentiation of B. cereus ad B. thuringiensis detected in food products could be a real contri- bution to the collection of epidemiological data. B. cereus, B. thuringiensis and B. anthracis have been subjected to several genomic studies to understand the taxo- nomic relationships. Data reported by Helgason et al. (2000a) reveal that B. cereus, B. thuringiensis and B. anthracis are indistinguishable using MEE or sequence analyses of nine genes, according with data obtained using DNA-DNA hybridization analysis by Kaneko et al. (1978) and Priest et al. (1994). Genetic evidence indicates that B. cereus, B. thuringiensis and B. anthracis are one s Cherif et al. (2003a) grouped B. anthracis in a separa pecies (Helgason et al. 2000b). te branch from B. cereus and B. thuringiensis on the basis of the phylogenetic relationship between the species of the B. cereus group using the long ITS containing t DNA. Data on ITS sequences confirm the difficulty in distinguishing B. cereus and B. thuringiensis apart from
ea ena Te one teen ere IR eee ee a a a ee ERS ae ee other Bacillus species (Table 3). The emetic type toxin is a 1.2 kDa molecular weight cereulide (ionosphoric water-soluble peptide) resistant to a treatment at 120°C for | h (heat-stable toxin) (Agata et al. 2002). This toxin may persist in foods after vegetative cells death. It causes an acute attack of vomiting after ingestion of contaminated food. For diar- rheal syndrome food containing more than 10* cells/spore per g has been considered risky, whereas for the emetic syndrome the number of B. cereus varies from 10° to 10° per g. Cereulide production begins at the end of the log growth phase. Outbreaks caused by B. cereus and/or the related species are often confused with out- breaks caused by other etiological agents. The symptoms of the diarrheal syndrome due to the presence of B. cereus are similar to the C. perfringens syndrome symp- toms. Symptoms due to B. cereus emetic toxins are similar to Staphylococcus intoxication symptoms. For this reason it is necessary an accurate laboratory investigation. Moreover B. cereus and B. thuringiensis are very closely related species belonging to the B. cereus group which main difference is the presence on B. thuringiensis plasmids of genes coding for the insecticidal toxins (Chen and Tsen 2002; Rasko et al. 2005). Plasmids loss and horizontal spreading of plasmids cause many difficulties in distinguishing B. thuringiensis from B. cereus affecting taxonomy and pathogenicity (Ward and Ellard 1983; Wilcks et al. 1998). Gonzales et al. (1982) used the high-frequency transfer of plasmids between strains of B. thuringiensis to study the genetic relarionship between plasmid and toxin produc- tion. Schnepf et al. (1998) worked on pesticidal proteins. Molecular methods have been developed to detect microbial toxins in foods. Radhika et al. (2002) utilized a probe to detect the phospholipase C. Virulence factors (enterotoxins, phospholi- pases and exotoxins) important for pathogenesis, were explored by the microarray- based detection by Sergeev et al. (2006).
ea ena Te one teen ere IR eee ee a a a ee ERS ae ee other Bacillus species (Table 3). The emetic type toxin is a 1.2 kDa molecular weight cereulide (ionosphoric water-soluble peptide) resistant to a treatment at 120°C for | h (heat-stable toxin) (Agata et al. 2002). This toxin may persist in foods after vegetative cells death. It causes an acute attack of vomiting after ingestion of contaminated food. For diar- rheal syndrome food containing more than 10* cells/spore per g has been considered risky, whereas for the emetic syndrome the number of B. cereus varies from 10° to 10° per g. Cereulide production begins at the end of the log growth phase. Outbreaks caused by B. cereus and/or the related species are often confused with out- breaks caused by other etiological agents. The symptoms of the diarrheal syndrome due to the presence of B. cereus are similar to the C. perfringens syndrome symp- toms. Symptoms due to B. cereus emetic toxins are similar to Staphylococcus intoxication symptoms. For this reason it is necessary an accurate laboratory investigation. Moreover B. cereus and B. thuringiensis are very closely related species belonging to the B. cereus group which main difference is the presence on B. thuringiensis plasmids of genes coding for the insecticidal toxins (Chen and Tsen 2002; Rasko et al. 2005). Plasmids loss and horizontal spreading of plasmids cause many difficulties in distinguishing B. thuringiensis from B. cereus affecting taxonomy and pathogenicity (Ward and Ellard 1983; Wilcks et al. 1998). Gonzales et al. (1982) used the high-frequency transfer of plasmids between strains of B. thuringiensis to study the genetic relarionship between plasmid and toxin produc- tion. Schnepf et al. (1998) worked on pesticidal proteins. Molecular methods have been developed to detect microbial toxins in foods. Radhika et al. (2002) utilized a probe to detect the phospholipase C. Virulence factors (enterotoxins, phospholi- pases and exotoxins) important for pathogenesis, were explored by the microarray- based detection by Sergeev et al. (2006).
Table 4 Characteristics used for differentiating Bacillus cereus group species +: 90-100% positive; — : 90-100% positive; +/—: 50-50% positive
Table 4 Characteristics used for differentiating Bacillus cereus group species +: 90-100% positive; — : 90-100% positive; +/—: 50-50% positive
strain level (Table 5). These molecular techniques can be used either on isolates from agar plate medium or on microbial DNA directly extracted from food. The need to obtain fast methods in food analyses stimulate research to optimize mo ecular protocols. Pulsed field gel electrophoresis (PFGE) and multilocus enzymes elecrophoresis are methods useful to analyze the genetic diversity of B. cereus and B. thuringiensis isolates (Helgason et al. 2000b). Fricker et al. (2007) proposed Real-Time PCR assays for the detection of emetic Bacillus cereus strains in foods. The advantage obtained in using Real-time PCR is the higly specificity, sensitivi fication (Mackay 2004). The assay targeted a specific part of the ce (ces) gene allowing the detection of the microorganism responsi ty and the quanti- reulide synthetase ble for the emetic food-poisoning outbreaks. The developed method was able to detect 10° CFUg"' after an enrichment time of 4-6 h, whereas the detection limit was 10'-103 CFUg"! directly form food (Fricker et al. 2007). Also a PCR method using colony hybrid- ization has been developed to detect B. cereus in foods by Radhika et al. (2002).
strain level (Table 5). These molecular techniques can be used either on isolates from agar plate medium or on microbial DNA directly extracted from food. The need to obtain fast methods in food analyses stimulate research to optimize mo ecular protocols. Pulsed field gel electrophoresis (PFGE) and multilocus enzymes elecrophoresis are methods useful to analyze the genetic diversity of B. cereus and B. thuringiensis isolates (Helgason et al. 2000b). Fricker et al. (2007) proposed Real-Time PCR assays for the detection of emetic Bacillus cereus strains in foods. The advantage obtained in using Real-time PCR is the higly specificity, sensitivi fication (Mackay 2004). The assay targeted a specific part of the ce (ces) gene allowing the detection of the microorganism responsi ty and the quanti- reulide synthetase ble for the emetic food-poisoning outbreaks. The developed method was able to detect 10° CFUg"' after an enrichment time of 4-6 h, whereas the detection limit was 10'-103 CFUg"! directly form food (Fricker et al. 2007). Also a PCR method using colony hybrid- ization has been developed to detect B. cereus in foods by Radhika et al. (2002).
Table 6 Microorganisms used to check primers BCFWI1 and BCREVNEW
Table 6 Microorganisms used to check primers BCFWI1 and BCREVNEW
Fig. 1 Amplicons obtained using different inocula of Bacillus cereus in the coffee concentrate samples (Manzano et al. 2003b). Lanes 1-7, samples artificially contaminated; lane 1, 1 x 10° cells mL7!; lane 2, 1 x 10° cells mL7!; lane 3, 1 x 10* cells mL7!; lane 4, 1 x 10° cells mL~!; lane 5, 1 x 10° cells mL~'; lane 6, 10 cells mL~'; lane 7, 1-10 cells mL“; lane 8, B. cereus DSMZ 2301 (positive control); lane 9, negative control; lane 10, (MWM) molecular weight marker (100 bp; Sigma, Germany)
Fig. 1 Amplicons obtained using different inocula of Bacillus cereus in the coffee concentrate samples (Manzano et al. 2003b). Lanes 1-7, samples artificially contaminated; lane 1, 1 x 10° cells mL7!; lane 2, 1 x 10° cells mL7!; lane 3, 1 x 10* cells mL7!; lane 4, 1 x 10° cells mL~!; lane 5, 1 x 10° cells mL~'; lane 6, 10 cells mL~'; lane 7, 1-10 cells mL“; lane 8, B. cereus DSMZ 2301 (positive control); lane 9, negative control; lane 10, (MWM) molecular weight marker (100 bp; Sigma, Germany)
Table 7 Bacillus strains used for PCR optimization in RE-PCR protocol
Table 7 Bacillus strains used for PCR optimization in RE-PCR protocol
Table 8 B. cereus/thuringiensis, tested
Table 8 B. cereus/thuringiensis, tested
Fig. 2 PCR products digested by Hinf I endonuclease. Lane 1: B. thuringiensis DSMZ 2046; Lane 2: B. thuringiensis from coffee; Lane 3: Bacillus thuringiensis DSMZ 350; Lane 4: Bacillus cereus DSMZ 4282; Lane 5: B. cereus from ricotta ; Lane 6: B. cereus DSMZ 2301; Lane 7: uncut PCR product B. cereus DSMZ 2301 ; Lane 8: Molecular Weight Marker, (Sigma, Milan, Italy)
Fig. 2 PCR products digested by Hinf I endonuclease. Lane 1: B. thuringiensis DSMZ 2046; Lane 2: B. thuringiensis from coffee; Lane 3: Bacillus thuringiensis DSMZ 350; Lane 4: Bacillus cereus DSMZ 4282; Lane 5: B. cereus from ricotta ; Lane 6: B. cereus DSMZ 2301; Lane 7: uncut PCR product B. cereus DSMZ 2301 ; Lane 8: Molecular Weight Marker, (Sigma, Milan, Italy)
Table 9 Identification results by the RE method (Hinf 1) on PCR product obtained by BCFW1 and BCRW1
Table 9 Identification results by the RE method (Hinf 1) on PCR product obtained by BCFW1 and BCRW1
Table 10 Strain distribution in clusters Primer REP 1R-Dt of 15 bp and the primer REP 2-Dt of 22 bp were used (Gevers et al. 2001). As reported in Fig. 2, ten clusters (A to L) were obtained by the rep-PCR fingerprints analysis using 80% similarity (Fig. 4). rep-PCR was more effective in clustering Bacillus strains. The technique clusters strains from the same origin as reported in cluster C, D, F, I and L. In cluster E are grouped strains from pesticides and in cluster C strains from patients (Table 11) (Manzano et al. 2009). According with Abriouel et al. (2007) “the utilization of conserved repetitive sequences dispersed in the whole genome is useful in contributing to the differen- tiation of genomic groups and check their association with foods”.
Table 10 Strain distribution in clusters Primer REP 1R-Dt of 15 bp and the primer REP 2-Dt of 22 bp were used (Gevers et al. 2001). As reported in Fig. 2, ten clusters (A to L) were obtained by the rep-PCR fingerprints analysis using 80% similarity (Fig. 4). rep-PCR was more effective in clustering Bacillus strains. The technique clusters strains from the same origin as reported in cluster C, D, F, I and L. In cluster E are grouped strains from pesticides and in cluster C strains from patients (Table 11) (Manzano et al. 2009). According with Abriouel et al. (2007) “the utilization of conserved repetitive sequences dispersed in the whole genome is useful in contributing to the differen- tiation of genomic groups and check their association with foods”.
Fig. 4 rep-PCR dendrogram of Bacillus cereus and B. thuringiensis strains determined by the Unweighted Pair Group Method using Arithmetic Average (UPGMA), correlation coefficient of 80% Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods
Fig. 4 rep-PCR dendrogram of Bacillus cereus and B. thuringiensis strains determined by the Unweighted Pair Group Method using Arithmetic Average (UPGMA), correlation coefficient of 80% Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods
to an increasingly denaturing environment partially melts till the completely dissociation into single strands at the reaching of extreme denaturing conditions. The position in the gel w here a DNA fragment subjected to denaturing gradient, melts and stops as migrating is dependent on the nucleotide sequence in the melted region. Different sequences stops at different positions (levels). DGGE/TTGE is a good method of finding po ymorphisms when DNA samples amplified from different individuals are run on denaturing gradient gels. This method has been applied in many protocols for identi et al. 2001; Ercolini 2004 BCRW1 were used for am fication or differentiation of strains from food (Vasquez ). Primers BCFW1 with a 40 bp GC clamp added, and amplicons from B. thuring plification of 69 amplicons from B. cereus strains and 15 iensis. Amplicons were used in a sequence-specific sepa- ration obtained by the TTGE technique. B. cereus amplicons were splitted in seven levels as reported in Table B. thuringiensis were 12. splitted in two levels: the first for seven strains from
to an increasingly denaturing environment partially melts till the completely dissociation into single strands at the reaching of extreme denaturing conditions. The position in the gel w here a DNA fragment subjected to denaturing gradient, melts and stops as migrating is dependent on the nucleotide sequence in the melted region. Different sequences stops at different positions (levels). DGGE/TTGE is a good method of finding po ymorphisms when DNA samples amplified from different individuals are run on denaturing gradient gels. This method has been applied in many protocols for identi et al. 2001; Ercolini 2004 BCRW1 were used for am fication or differentiation of strains from food (Vasquez ). Primers BCFW1 with a 40 bp GC clamp added, and amplicons from B. thuring plification of 69 amplicons from B. cereus strains and 15 iensis. Amplicons were used in a sequence-specific sepa- ration obtained by the TTGE technique. B. cereus amplicons were splitted in seven levels as reported in Table B. thuringiensis were 12. splitted in two levels: the first for seven strains from
Table 12 Levels obtained by PCR-TTGE analyses applied to B. cereus strains present in this microbial group (Sayama and Bork 2001; Rasko et al. 2005). These regions can be the result of changes due to the insertion or deletion of mobile elements. Also utilizing molecular methods it is difficult to identify a Bacillus strain as the genetic exchange between Bacillus strains, in the absence of genetic barriers, rules out this kind of classification at the species and subspecies level.
Table 12 Levels obtained by PCR-TTGE analyses applied to B. cereus strains present in this microbial group (Sayama and Bork 2001; Rasko et al. 2005). These regions can be the result of changes due to the insertion or deletion of mobile elements. Also utilizing molecular methods it is difficult to identify a Bacillus strain as the genetic exchange between Bacillus strains, in the absence of genetic barriers, rules out this kind of classification at the species and subspecies level.
Fig. 1 Slices of dry cured ham and pig skin
Fig. 1 Slices of dry cured ham and pig skin
Fig. 2. Box and ionizer system 40 DCH and 40 PS samples were inoculated. 10 ml of water (control samples) or 10 ml of three different solutions of hydrogen peroxide (20%, 10%, and 3%) were added to the samples (ten each). The samples were split in two groups. One group was kept in air, the second group was packaged under vacuum in PET. After 10 min
Fig. 2. Box and ionizer system 40 DCH and 40 PS samples were inoculated. 10 ml of water (control samples) or 10 ml of three different solutions of hydrogen peroxide (20%, 10%, and 3%) were added to the samples (ten each). The samples were split in two groups. One group was kept in air, the second group was packaged under vacuum in PET. After 10 min
Fig. 3_ Behaviour of Listeria monocytogenes in dry cured ham packaged in MAP at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample
Fig. 3_ Behaviour of Listeria monocytogenes in dry cured ham packaged in MAP at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample
Fig. 4 Behaviour of Listeria monocytogenes in dry cured ham packaged under vacuum at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample
Fig. 4 Behaviour of Listeria monocytogenes in dry cured ham packaged under vacuum at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample
Fig. 5 Behaviour of L. monocytogenes in pig skin treated with ionized air. Legend: NT: not treated; T: treated
Fig. 5 Behaviour of L. monocytogenes in pig skin treated with ionized air. Legend: NT: not treated; T: treated
Fig. 6 Behaviour of L. monocytogenes in dry cured ham treated with ionized air. Legend: NT: not treated; T: treated
Fig. 6 Behaviour of L. monocytogenes in dry cured ham treated with ionized air. Legend: NT: not treated; T: treated
Table 1 Behaviour of L. monocytogenes in dry cured ham treated with ozonated water * Mean values with different letters are statistically different (P < 0.001) Table 2, Behaviour of L. monocytogenes in pig skin treated with ozonated water
Table 1 Behaviour of L. monocytogenes in dry cured ham treated with ozonated water * Mean values with different letters are statistically different (P < 0.001) Table 2, Behaviour of L. monocytogenes in pig skin treated with ozonated water
« Mean values with different letters are statistically different (P < 0.001) mut the same decrease was observed in the control, where the samples were treated vith water without added ozone. The samples obtained with water without added yzone showed a lower concentration of L. monocytogenes than the untreated sam- les. Indeed, viable L. monocytogenes was found in ozonized water obtained from he treated samples. Significant differences (P < 0.001) were observed when com- yaring the treated (with or without added ozone) and the untreated samples. Conversely, there are no significant differences between the samples treated with ind without added ozone. Therefore, the decrease might be due to the mechanical iction (washing) of water on the samples. The lack activity of the ozonized water night be due to the water temperature (15°C) and to a low ozone concentration in water (0.2 ppm). The ozone generator that we used did not allow to cool water lown to 4°C or to increase the ozone concentration. The bactericidal activity of yzonized water could be improved if water at 4°C was added (Kaothien et al. 2001) ind if its concentration were equal to or higher than 20 ppm (Wade et al. 2003). Indeed, the matrix (DCH or PS) played a role in the ineffectiveness of the treat- nent. The presence of organic compounds in the matrix may degrade the ozone and “onsequently its oxidative activity (Moore et al. 2000). For this reason, Beltran
« Mean values with different letters are statistically different (P < 0.001) mut the same decrease was observed in the control, where the samples were treated vith water without added ozone. The samples obtained with water without added yzone showed a lower concentration of L. monocytogenes than the untreated sam- les. Indeed, viable L. monocytogenes was found in ozonized water obtained from he treated samples. Significant differences (P < 0.001) were observed when com- yaring the treated (with or without added ozone) and the untreated samples. Conversely, there are no significant differences between the samples treated with ind without added ozone. Therefore, the decrease might be due to the mechanical iction (washing) of water on the samples. The lack activity of the ozonized water night be due to the water temperature (15°C) and to a low ozone concentration in water (0.2 ppm). The ozone generator that we used did not allow to cool water lown to 4°C or to increase the ozone concentration. The bactericidal activity of yzonized water could be improved if water at 4°C was added (Kaothien et al. 2001) ind if its concentration were equal to or higher than 20 ppm (Wade et al. 2003). Indeed, the matrix (DCH or PS) played a role in the ineffectiveness of the treat- nent. The presence of organic compounds in the matrix may degrade the ozone and “onsequently its oxidative activity (Moore et al. 2000). For this reason, Beltran
Table 3. Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept in air
Table 3. Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept in air
Table 4 Behaviour of Listeria monocytogenes in pig skin treated with different concentrations of H,O, and kept in air
Table 4 Behaviour of Listeria monocytogenes in pig skin treated with different concentrations of H,O, and kept in air
Table 5 Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept under vacuum
Table 5 Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept under vacuum
3% (Table 5) hydrogen peroxide solution respectively in PS kept in air and pack- aged under vacuum.
3% (Table 5) hydrogen peroxide solution respectively in PS kept in air and pack- aged under vacuum.
Fig. 7 (a) Dry cured ham treated with H,O, (20% v/v) (b) Dry cured ham treated with H,O, (10% v/v) (c) Dry cured ham treated with H,O, (3% v/v)
Fig. 7 (a) Dry cured ham treated with H,O, (20% v/v) (b) Dry cured ham treated with H,O, (10% v/v) (c) Dry cured ham treated with H,O, (3% v/v)
Table 7 Behaviour of Listeria monocytogenes in DCH and PS treated with different mixes of essential oil Legend: DCH: dry cured ham; PS: Pig skin; A: solution A B: Solution B; C: solution (mix A + B + water) X: average; SD: standard deviation.
Table 7 Behaviour of Listeria monocytogenes in DCH and PS treated with different mixes of essential oil Legend: DCH: dry cured ham; PS: Pig skin; A: solution A B: Solution B; C: solution (mix A + B + water) X: average; SD: standard deviation.
Table 8 Behaviour of Leuconostoc carnosum CH versus Listeria monocytogenes in Data: UFC/cm?
Table 8 Behaviour of Leuconostoc carnosum CH versus Listeria monocytogenes in Data: UFC/cm?
Other sources of infection include contaminated surgical instruments anc donor organs. Health care workers, dentists, and others who have frequent contac with infected blood or blood products are at highest risk. HBV transmissior from infected healthcare personnel to patients is relatively uncommon, but ha: occurred during invasive surgical, obstetrical, or dental procedures. Renal dialysi: mentioned by Alter et al. (1983), and long-term household or intimate non-sexua contact with an HBsAg-positive individual also exposes people to the risk o: acquiring HBV unless necessary precautions have been taken beforehand (HBV vaccination, using separate dialysis equipment etc.). Binallyvy came “trendv”’ annlicatione ench ac tattqninga ear/hodyv niercino o
Other sources of infection include contaminated surgical instruments anc donor organs. Health care workers, dentists, and others who have frequent contac with infected blood or blood products are at highest risk. HBV transmissior from infected healthcare personnel to patients is relatively uncommon, but ha: occurred during invasive surgical, obstetrical, or dental procedures. Renal dialysi: mentioned by Alter et al. (1983), and long-term household or intimate non-sexua contact with an HBsAg-positive individual also exposes people to the risk o: acquiring HBV unless necessary precautions have been taken beforehand (HBV vaccination, using separate dialysis equipment etc.). Binallyvy came “trendv”’ annlicatione ench ac tattqninga ear/hodyv niercino o
Fig. 1 Geographical distribution of HEV isolates according to genotypes (Gt). HEV Gt | and 2: epidemic strains causing human infection. HEV Gt 3 and 4: zoonotic strains isolated from humans and a variety of animals, particularly pigs. In some countries, different genotypes co-circulate in distinct ecological niches: Gt 1 and 4 in China, India, and Vietnam; Gt | and 2 in several African countries, including Namibia, Chad, and Sudan; Gt 3 and 4 in Japan; Gt | and 3 in Cambodia; Gt 2 and 3 in Mexico (www.eht-forum.org) death rate during pregnancy approaches 15-25% with possibilities of foetal death, abortion, premature delivery, or death of a live-born baby soon after birth (Smith 2001; Chibber et al. 2004). HEV is transmitted primarily by the faecal-oral route and waterborne epidemics are characteristics of hepatitis E. HEV, known for being the cause of major outbreaks of waterborne hepatitis in Asia and Africa, is an emerging pathogen in industrialized countries (Bihl and Negro 2008). Clinical cases of HEV infection in developed countries have generally been associated with travel to tropical or subtropical countries. There has also been an increasing number of reports from Europe and the United States of sporadic hepatitis attributable to HEV but not associated with travel, leading to suggestions that HEV may be endemic at low levels in developed countries. HEV has been reported in Netherlands (Widdowson et al. 2003), Spain (Peralta et al. 2009), France (Legrand-Abravanel et al. 2009), Greece (Stefanidis et al. 2004), Sweden (Olsen et al. 2006), UK (Ijaz et al. 2009), Italy (Di Bartolo et al. 2008), United States (Atiq et al. 2009), Japan (Tanaka et al. 2001) more cases are likely to be reported (Fig. 1). It has been recognised that HEV and HEV related viruses circulate in domestic animals native to industrial countries and should be considered as a potential
Fig. 1 Geographical distribution of HEV isolates according to genotypes (Gt). HEV Gt | and 2: epidemic strains causing human infection. HEV Gt 3 and 4: zoonotic strains isolated from humans and a variety of animals, particularly pigs. In some countries, different genotypes co-circulate in distinct ecological niches: Gt 1 and 4 in China, India, and Vietnam; Gt | and 2 in several African countries, including Namibia, Chad, and Sudan; Gt 3 and 4 in Japan; Gt | and 3 in Cambodia; Gt 2 and 3 in Mexico (www.eht-forum.org) death rate during pregnancy approaches 15-25% with possibilities of foetal death, abortion, premature delivery, or death of a live-born baby soon after birth (Smith 2001; Chibber et al. 2004). HEV is transmitted primarily by the faecal-oral route and waterborne epidemics are characteristics of hepatitis E. HEV, known for being the cause of major outbreaks of waterborne hepatitis in Asia and Africa, is an emerging pathogen in industrialized countries (Bihl and Negro 2008). Clinical cases of HEV infection in developed countries have generally been associated with travel to tropical or subtropical countries. There has also been an increasing number of reports from Europe and the United States of sporadic hepatitis attributable to HEV but not associated with travel, leading to suggestions that HEV may be endemic at low levels in developed countries. HEV has been reported in Netherlands (Widdowson et al. 2003), Spain (Peralta et al. 2009), France (Legrand-Abravanel et al. 2009), Greece (Stefanidis et al. 2004), Sweden (Olsen et al. 2006), UK (Ijaz et al. 2009), Italy (Di Bartolo et al. 2008), United States (Atiq et al. 2009), Japan (Tanaka et al. 2001) more cases are likely to be reported (Fig. 1). It has been recognised that HEV and HEV related viruses circulate in domestic animals native to industrial countries and should be considered as a potential
HEV is the major etiologic agent of enterically transmitted non-A, non-B hepatitis worldwide. It is a spherical, non-enveloped, single (plus) stranded RNA virus that is approximately 32-34 nm in diameter (Fig. 2). HEV belongs to a genus of HEV-like viruses (unassigned genus). Hepatitis E virus (HEV) is a positive- stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified: the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest it is more closely related to the Togaviridae (Yang and Vyas 1996).
HEV is the major etiologic agent of enterically transmitted non-A, non-B hepatitis worldwide. It is a spherical, non-enveloped, single (plus) stranded RNA virus that is approximately 32-34 nm in diameter (Fig. 2). HEV belongs to a genus of HEV-like viruses (unassigned genus). Hepatitis E virus (HEV) is a positive- stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified: the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest it is more closely related to the Togaviridae (Yang and Vyas 1996).
Fig. 3. Hepatitis E virus, complete genome (http://www.ncbi.nlm.nih.gov/nuccore/9626440?repo rt=graph&from=1&to=7105) The incubation period following exposure to HEV ranges from 3 to 8 weeks, with a mean of 40 days. The period of communicability is unknown. There are no chronic infections reported. However, recent reports present biochemical, histological and genetic evidence of chronic HEV infection in transplant patients (reviewed by Chandra et al. 2008). 4° oe 4 . . 44 er ate: a
Fig. 3. Hepatitis E virus, complete genome (http://www.ncbi.nlm.nih.gov/nuccore/9626440?repo rt=graph&from=1&to=7105) The incubation period following exposure to HEV ranges from 3 to 8 weeks, with a mean of 40 days. The period of communicability is unknown. There are no chronic infections reported. However, recent reports present biochemical, histological and genetic evidence of chronic HEV infection in transplant patients (reviewed by Chandra et al. 2008). 4° oe 4 . . 44 er ate: a
Fig. 4 Genetic relatedness between HEV strains from human and non-human sources and from various countries. Minimal spanning trees of 81 sequences of 148 nucleotides HEV RNA showing genetic distances between genotype three HEV strains from humans and animals. (a) Strains labelled by geographical origin. (b) Strains labelled by biological origin. The 13 recent Dutch cases are marked with ID numbers inside the coloured circles (adapted from Borgen et al. BMC Infectious Diseases 2008 8:61 doi:10.1186/1471-2334-8-61)
Fig. 4 Genetic relatedness between HEV strains from human and non-human sources and from various countries. Minimal spanning trees of 81 sequences of 148 nucleotides HEV RNA showing genetic distances between genotype three HEV strains from humans and animals. (a) Strains labelled by geographical origin. (b) Strains labelled by biological origin. The 13 recent Dutch cases are marked with ID numbers inside the coloured circles (adapted from Borgen et al. BMC Infectious Diseases 2008 8:61 doi:10.1186/1471-2334-8-61)
Table 1 Advantages and disadvantages of the main techniques used by laboratories in serological and virological dengue diagnostic assays
Table 1 Advantages and disadvantages of the main techniques used by laboratories in serological and virological dengue diagnostic assays
Table 2. Examples of commercial kits for DENV diagnostics
Table 2. Examples of commercial kits for DENV diagnostics
Fig. 1 Descriptive scheme of the experimental setups for dengue virus detection. (A) Photon counting unit. (Al) Hamamatsu HC135-01 PMT Sensor Module. (A2) PMT fixation ring. (A3) Manual shutter (71430, Oriel). (A4) Fiber holder that prevents the movement of the fiber inside the photon counting unit. (A5) Fiber optic. (A6) Connection wire of PMT to computer. (A7) Electricity cable. (B) The outside handle of manual shutter that enables light access to the PMT. (C) Immobilization unit. (C1) Fiber optic. (C2) 100ul pipette tip. (C3) Conical tube cup. (C4) Point of fixation of fiber. (C5) Optical fiber core. (C6) Biorecognition elements according to MAC-ELISA chemiluminescent OFIS (Atias et al. 2009). (C7) Test samples. (E) Connection to computer
Fig. 1 Descriptive scheme of the experimental setups for dengue virus detection. (A) Photon counting unit. (Al) Hamamatsu HC135-01 PMT Sensor Module. (A2) PMT fixation ring. (A3) Manual shutter (71430, Oriel). (A4) Fiber holder that prevents the movement of the fiber inside the photon counting unit. (A5) Fiber optic. (A6) Connection wire of PMT to computer. (A7) Electricity cable. (B) The outside handle of manual shutter that enables light access to the PMT. (C) Immobilization unit. (C1) Fiber optic. (C2) 100ul pipette tip. (C3) Conical tube cup. (C4) Point of fixation of fiber. (C5) Optical fiber core. (C6) Biorecognition elements according to MAC-ELISA chemiluminescent OFIS (Atias et al. 2009). (C7) Test samples. (E) Connection to computer
Table 3 Comparison of the chemiluminescent OFIS method to the chemiluminescent and colorimetric MAC-ELISA assays. (Atias et al. 2009) ising chemiluminescent OFIS (Atias et al. 2009). Since IgM is a much large nolecule than IgG, it is difficult to work with in chemiluminescent OFIS, as the ussay is based on bottom to top covalent binding and top to bottom detection but 1ot adsorption. However, the main serological routine test in dengue endemic -ountries is based on anti-DENV IgM detection, so it is important to focus on this <ind of diagnostic.
Table 3 Comparison of the chemiluminescent OFIS method to the chemiluminescent and colorimetric MAC-ELISA assays. (Atias et al. 2009) ising chemiluminescent OFIS (Atias et al. 2009). Since IgM is a much large nolecule than IgG, it is difficult to work with in chemiluminescent OFIS, as the ussay is based on bottom to top covalent binding and top to bottom detection but 1ot adsorption. However, the main serological routine test in dengue endemic -ountries is based on anti-DENV IgM detection, so it is important to focus on this <ind of diagnostic.
Table 1 (continued) “The term ‘microparasite’ was used in the sense of Anderson and May (1998): small-sized parasites able to replicate usually at very high rates within the host In the context of the present work, microparasites are exclusively eukaryotic microorganisms (protists and fungi). Most data come from Woolhouse et a (2006) and Fagherazzi-Pagel (2009).
Table 1 (continued) “The term ‘microparasite’ was used in the sense of Anderson and May (1998): small-sized parasites able to replicate usually at very high rates within the host In the context of the present work, microparasites are exclusively eukaryotic microorganisms (protists and fungi). Most data come from Woolhouse et a (2006) and Fagherazzi-Pagel (2009).
Fig. 1 Development of Cryptosporidium muris in the SCID mouse. Stomach section from a SCID mouse euthanatized 46 days post-infection. Gastric glan are filled with numerous parasites at different developmental stages. Hematoxylin & Eosin staining. Bar = 10 um
Fig. 1 Development of Cryptosporidium muris in the SCID mouse. Stomach section from a SCID mouse euthanatized 46 days post-infection. Gastric glan are filled with numerous parasites at different developmental stages. Hematoxylin & Eosin staining. Bar = 10 um
‘Data from AFSSA (2002), Millar et al. (2002), Dawson (2005) and Smith et al. (2007) Table 3. Reported cases of foodborne cryptosporidiosis (non-exhaustive list)*
‘Data from AFSSA (2002), Millar et al. (2002), Dawson (2005) and Smith et al. (2007) Table 3. Reported cases of foodborne cryptosporidiosis (non-exhaustive list)*
Table 4 Emerging and reemerging foodborne helminthes*
Table 4 Emerging and reemerging foodborne helminthes*
* Most data come from Woolhouse et al. (2006) and Fagherazzi-Pagel (2009)
* Most data come from Woolhouse et al. (2006) and Fagherazzi-Pagel (2009)
Table 1 Specific activities of selected enzymes involved in thymidylate de novo biosynthesis in T. spiralis and T: pseudospiralis muscle larvae, T. spiralis adult forms, C. elegans adult forms, and L1, L3 and dauer larvae. For both Trichinella species time between infection and isolation is indicated
Table 1 Specific activities of selected enzymes involved in thymidylate de novo biosynthesis in T. spiralis and T: pseudospiralis muscle larvae, T. spiralis adult forms, C. elegans adult forms, and L1, L3 and dauer larvae. For both Trichinella species time between infection and isolation is indicated
Fig. 1 (a) Thymidylate synthase and histone H.3-like mRNA, and 18S rRNA, bands in T. spira- lis muscle larvae (ML), adult forms (A) and newborn larvae (NBL). (b) Thymidylate synthase mRNA level in T.spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. (c) Histone H.3-like mRNA level in T:spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. Densitometric results in B and C are presented as mean result of three separate experiments + SD for ML and (A), and as mean result of two separate experiments for NBL + % difference from each of the two results.
Fig. 1 (a) Thymidylate synthase and histone H.3-like mRNA, and 18S rRNA, bands in T. spira- lis muscle larvae (ML), adult forms (A) and newborn larvae (NBL). (b) Thymidylate synthase mRNA level in T.spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. (c) Histone H.3-like mRNA level in T:spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. Densitometric results in B and C are presented as mean result of three separate experiments + SD for ML and (A), and as mean result of two separate experiments for NBL + % difference from each of the two results.
Fig. 2. Immunoblot analysis of cross-reactivity of monoclonal antibody generated against rat hepatoma thymidylate synthase. T: spiralis muscle larvae crude extract (lane 1; 100 tg protein), purified recombinant (lane 2; 1 jg protein) or purified muscle larvae endogenous (lane 3; 1 ug protein) thymidylate synthase, rat purified regenerating liver endogenous (lane 4; | Lg protein) or rat recombinant (lane 5; 1 ug protein) thymidylate synthase and L5178Y crude extract (lane 6; 100 ig protein) were separated by SDS polyacrylamide (12%) gel electrophoresis and proteins were transferred into PVDF membrane. Thymidylate synthase was detected by treatment with mono- clonal antibody and goat anti-mouse IgG (H + L) — HRP conjugate (BioRad) as a secondary antibody
Fig. 2. Immunoblot analysis of cross-reactivity of monoclonal antibody generated against rat hepatoma thymidylate synthase. T: spiralis muscle larvae crude extract (lane 1; 100 tg protein), purified recombinant (lane 2; 1 jg protein) or purified muscle larvae endogenous (lane 3; 1 ug protein) thymidylate synthase, rat purified regenerating liver endogenous (lane 4; | Lg protein) or rat recombinant (lane 5; 1 ug protein) thymidylate synthase and L5178Y crude extract (lane 6; 100 ig protein) were separated by SDS polyacrylamide (12%) gel electrophoresis and proteins were transferred into PVDF membrane. Thymidylate synthase was detected by treatment with mono- clonal antibody and goat anti-mouse IgG (H + L) — HRP conjugate (BioRad) as a secondary antibody
Table 1 Major parasitic helminths of humans
Table 1 Major parasitic helminths of humans
Table 2 Helminth* products modulating host immune response
Table 2 Helminth* products modulating host immune response
Fig. 1 Effect of HP-NAP treatment on plasma levels of total and TsE/S-specific IgE. In control animals (open squares) TsE/S-specific IgE levels were high up to day 42, whereas in HP-NAP- treated animals (solid squares) IgE levels progressively decreased after day 14. Results are pre- sented as means + SD in 2 groups of 45 animals. **P < .0005 and ***P < .0001
Fig. 1 Effect of HP-NAP treatment on plasma levels of total and TsE/S-specific IgE. In control animals (open squares) TsE/S-specific IgE levels were high up to day 42, whereas in HP-NAP- treated animals (solid squares) IgE levels progressively decreased after day 14. Results are pre- sented as means + SD in 2 groups of 45 animals. **P < .0005 and ***P < .0001
Fig. 2. Effect of HP-NAP treatment on blood Th2 and Th1 parameters. Eosinophils and total IgE, IL-4, IL-5, IFN-y, and IL-12 levels were measured in the blood of control animals (open squares) and compared with values in animals treated on days 10 and 28 with HP-NAP (solid squares). Results are presented as means + SD
Fig. 2. Effect of HP-NAP treatment on blood Th2 and Th1 parameters. Eosinophils and total IgE, IL-4, IL-5, IFN-y, and IL-12 levels were measured in the blood of control animals (open squares) and compared with values in animals treated on days 10 and 28 with HP-NAP (solid squares). Results are presented as means + SD
Fig. 3 Experimental protocol of the study. HP-NAP has been delivered systemically (i.p.) (a) or via mucosal route (b). Groups of C57BL/6j mice were treated with saline, with OVA alone, with OVA plus i.p. HP-NAP or with OVA plus mucosal HP-NAP. In both systemic and mucosal proto- cols, mice were treated with OVA according to a procedure consisting of a first phase of sensitiza- tion with OVA i.p. (100 j1g/mouse) and a second phase of induction of the allergic response with aerosolized OVA (2% in PBS) for 5 min on day 8, and finally exposed to aerosolized antigen (1% in PBS) for 20 min daily on days 15-18. Control animals, designed as saline, were injected with PBS alone and then exposed to aerosolized PBS. In the systemic protocol (a) mice were treated with ip. HP-NAP (10 j1g/mouse) on day 1, whereas in the mucosal protocol (b) mice received intra-nasal HP-NAP (10 ug/mouse) on days 7 and 8
Fig. 3 Experimental protocol of the study. HP-NAP has been delivered systemically (i.p.) (a) or via mucosal route (b). Groups of C57BL/6j mice were treated with saline, with OVA alone, with OVA plus i.p. HP-NAP or with OVA plus mucosal HP-NAP. In both systemic and mucosal proto- cols, mice were treated with OVA according to a procedure consisting of a first phase of sensitiza- tion with OVA i.p. (100 j1g/mouse) and a second phase of induction of the allergic response with aerosolized OVA (2% in PBS) for 5 min on day 8, and finally exposed to aerosolized antigen (1% in PBS) for 20 min daily on days 15-18. Control animals, designed as saline, were injected with PBS alone and then exposed to aerosolized PBS. In the systemic protocol (a) mice were treated with ip. HP-NAP (10 j1g/mouse) on day 1, whereas in the mucosal protocol (b) mice received intra-nasal HP-NAP (10 ug/mouse) on days 7 and 8
On day 18, blood was taken from saline-, OVA- and OVA plus Systemic (Sy) HP-NAP-treated animals. Blood smears were stained to calculate the proportions and the mean (+ SE) absolute counts of lymphocytes, neutrophils, and eosinophils. Levels of total serum IgE were assessed by a specific ELISA assay. ** P < 0.01, *** P < 0.03 OVA plus Sy HP-NAP versus OVA alone. Table 3. HP-NAP delivery reduces serum IgE levels and eosinophil count in the blood of OVA eencitizved mire
On day 18, blood was taken from saline-, OVA- and OVA plus Systemic (Sy) HP-NAP-treated animals. Blood smears were stained to calculate the proportions and the mean (+ SE) absolute counts of lymphocytes, neutrophils, and eosinophils. Levels of total serum IgE were assessed by a specific ELISA assay. ** P < 0.01, *** P < 0.03 OVA plus Sy HP-NAP versus OVA alone. Table 3. HP-NAP delivery reduces serum IgE levels and eosinophil count in the blood of OVA eencitizved mire
Fig. 4 Intraperitoneal administration of HP-NAP inhibited the development of airway eosinophilia in OVA-sensitized animals. (a) On day 1, mice were sensitized with OVA alone or with OVA plus sys- temic HP-NAP (Sy HP-NAP), and then exposed to aerosolized OVA. Control animals were injected with PBS alone and then exposed to aerosolized PBS. Cytocentrifuge preparations from BAL of the three groups of animals on day 18 were stained to calculate the proportions of eosinophils, mac- rophages, neutrophils and lymphocytes. Absolute counts (+ SE) for each cell type were calculated from the number of total cells in the BAL. *** P < 0.01 versus OVA-treated mice. (b) Frozen sections of lungs from OVA-sensitized (eft), and Sy HP-NAP-treated OVA-sensitized animals (right). Eosinophil infiltration in the lung is virtually absent in OVA-sensitized mice treated with Sy HP-NAP Immunodulation and Helminths: Towards New Strategies for Treatment
Fig. 4 Intraperitoneal administration of HP-NAP inhibited the development of airway eosinophilia in OVA-sensitized animals. (a) On day 1, mice were sensitized with OVA alone or with OVA plus sys- temic HP-NAP (Sy HP-NAP), and then exposed to aerosolized OVA. Control animals were injected with PBS alone and then exposed to aerosolized PBS. Cytocentrifuge preparations from BAL of the three groups of animals on day 18 were stained to calculate the proportions of eosinophils, mac- rophages, neutrophils and lymphocytes. Absolute counts (+ SE) for each cell type were calculated from the number of total cells in the BAL. *** P < 0.01 versus OVA-treated mice. (b) Frozen sections of lungs from OVA-sensitized (eft), and Sy HP-NAP-treated OVA-sensitized animals (right). Eosinophil infiltration in the lung is virtually absent in OVA-sensitized mice treated with Sy HP-NAP Immunodulation and Helminths: Towards New Strategies for Treatment
Table 4 HP-NAP delivery reduces the Th2 cytokine release in the bronchoalveolar lavage of OVA sensitized mice On day 18, bronchoalveolar lavage samples were collected from OVA- and OVA plus Systemic (Sy) or mucosal (Mu) HP-NAP-treated OVA-sensitized mice. Cytokines were assayed in the supernatants. Mean values (+ SE) are reported. * P < 0.05, ** P < 0.04 OVA plus Sy or Mu HP-NAP versus OVA alone mice.
Table 4 HP-NAP delivery reduces the Th2 cytokine release in the bronchoalveolar lavage of OVA sensitized mice On day 18, bronchoalveolar lavage samples were collected from OVA- and OVA plus Systemic (Sy) or mucosal (Mu) HP-NAP-treated OVA-sensitized mice. Cytokines were assayed in the supernatants. Mean values (+ SE) are reported. * P < 0.05, ** P < 0.04 OVA plus Sy or Mu HP-NAP versus OVA alone mice.
The “threshold effect” (concentration above which foods become unusable) of aflatoxin B1 is 20 ug L" of the total content of aflatoxins. AFB, is formed when the temperature is 40-50°. Entering the organism through fodder it is metabolized to aflatoxin M, that is given off into the milk. The threshold of AFM, content in milk is 5 pg L™'. For the maze used to feed milk cows this level is 20 ug L", for calves used to produce meat and pigs it is 300 ug L™ and for breeding stock — 100 ug L”. The allowable quantities of aflatoxin B, in cotton seeds is 300 wg L™'. An important condition for the toxic and carcinogenic effects of AFB, to be active is its conversion into a number of metabolites (Murphy et al. 2006). The aflatoxins possess the following biological activity. They are the strongest carcinogenic substances in nature. They are also strongly toxic. They suppress the activity of the immune system. They possess an important biological specificity. They have a cumulative effect — manifested by evacuation through the liver, urine and faeces, a peak moment occurs, that is a carcinoma is formed in it, which is due to the accumulation of a certain quantity of aflatoxin. It has been proven that there is a connection between the virus causing hepatitis B and the occurrence of liver cancer when aflatoxins are taken in. These mycotoxins posses tetragenic effect (cause malformations) of the fetus when the mother has taken contaminated food that penetrates the fetus through the umbilical cord. Thus they cause irreversible deformations such as imbecility, defects in the heart valves, only one ear, no fingers or one hand shorter than the other (Vlahov and Ivanon 1996). Of all the known mycotoxins aflatoxin B1 (AFB1) is the major and best studied one because it possesses exceptional toxicity and is widespread in market foods and
The “threshold effect” (concentration above which foods become unusable) of aflatoxin B1 is 20 ug L" of the total content of aflatoxins. AFB, is formed when the temperature is 40-50°. Entering the organism through fodder it is metabolized to aflatoxin M, that is given off into the milk. The threshold of AFM, content in milk is 5 pg L™'. For the maze used to feed milk cows this level is 20 ug L", for calves used to produce meat and pigs it is 300 ug L™ and for breeding stock — 100 ug L”. The allowable quantities of aflatoxin B, in cotton seeds is 300 wg L™'. An important condition for the toxic and carcinogenic effects of AFB, to be active is its conversion into a number of metabolites (Murphy et al. 2006). The aflatoxins possess the following biological activity. They are the strongest carcinogenic substances in nature. They are also strongly toxic. They suppress the activity of the immune system. They possess an important biological specificity. They have a cumulative effect — manifested by evacuation through the liver, urine and faeces, a peak moment occurs, that is a carcinoma is formed in it, which is due to the accumulation of a certain quantity of aflatoxin. It has been proven that there is a connection between the virus causing hepatitis B and the occurrence of liver cancer when aflatoxins are taken in. These mycotoxins posses tetragenic effect (cause malformations) of the fetus when the mother has taken contaminated food that penetrates the fetus through the umbilical cord. Thus they cause irreversible deformations such as imbecility, defects in the heart valves, only one ear, no fingers or one hand shorter than the other (Vlahov and Ivanon 1996). Of all the known mycotoxins aflatoxin B1 (AFB1) is the major and best studied one because it possesses exceptional toxicity and is widespread in market foods and
These mycotoxins are produced by fungi pathogenic for maze like Fusarium verticiliodes and Fusarium proliferetum and to a very small extent Alternaria in tomatoes. At least 15 varieties of fumosinins have been identified. They are readily dissolved in water and are distinguished from the other mycotoxins by their lack of aromatic structure or a unique chromophore, which does not allow easy analytic determination. Foods containing maze are the main hazard of contamination with fumosinins in the food industry (Vlahov and Ivanon 1996). Most of the foods on the market contain fumosinins to 500 ng g™ or less thanks to the control exercised on the maze and the other components. Fumosinin B, is the most toxic representa- tive of the group.
These mycotoxins are produced by fungi pathogenic for maze like Fusarium verticiliodes and Fusarium proliferetum and to a very small extent Alternaria in tomatoes. At least 15 varieties of fumosinins have been identified. They are readily dissolved in water and are distinguished from the other mycotoxins by their lack of aromatic structure or a unique chromophore, which does not allow easy analytic determination. Foods containing maze are the main hazard of contamination with fumosinins in the food industry (Vlahov and Ivanon 1996). Most of the foods on the market contain fumosinins to 500 ng g™ or less thanks to the control exercised on the maze and the other components. Fumosinin B, is the most toxic representa- tive of the group.
Fig. 3_ Normalized excitation and fluorescence spectra of membrane Magnetic nanoparticles as immobilization materials have the following advantages: (1) more specific surface area obtained for the binding of larger amounts of biomolecules; (2) lower mass transfer resistance; (3) selective separation of the immobilized biomolecules from a reaction mixture on application of a magnetic field. Among these materials, Fe,O,, magnetic nanoparticles are the most commonly studied. Fe,O,, magnetic nanoparticles have good biocompatibility, strong superpara- magnetism, low toxicity, and an easy preparation process, and their use in biosensors has already shown attractive prospects (Sheng-Fu Wang and Tan Yu-Mei 2007). Horseradish peroxidase (HRP) is an important peroxidase that contains heme,
Fig. 3_ Normalized excitation and fluorescence spectra of membrane Magnetic nanoparticles as immobilization materials have the following advantages: (1) more specific surface area obtained for the binding of larger amounts of biomolecules; (2) lower mass transfer resistance; (3) selective separation of the immobilized biomolecules from a reaction mixture on application of a magnetic field. Among these materials, Fe,O,, magnetic nanoparticles are the most commonly studied. Fe,O,, magnetic nanoparticles have good biocompatibility, strong superpara- magnetism, low toxicity, and an easy preparation process, and their use in biosensors has already shown attractive prospects (Sheng-Fu Wang and Tan Yu-Mei 2007). Horseradish peroxidase (HRP) is an important peroxidase that contains heme,
as a quantifiable o ptical signal. Optical biosensors offer the advantages of noninvasive nondestructive, continuous, and simultaneous multianalyte detection (Taitt et al 2005). Optical systems do not interfere with metabolism, and thus in vivo measure- ments are possib e. In this chapter, some applications of the following optical sensing principles in foodborne pathogen detection are discussed: (1) SPR, (2 resonance mirror (RM), (3) fiber-optic biosensor, (4) array biosensor, (5) Ramar spectroscopy, and (6) light-addressable potentiometric sensor (LAPS) (Knopk et al. 2007c).
as a quantifiable o ptical signal. Optical biosensors offer the advantages of noninvasive nondestructive, continuous, and simultaneous multianalyte detection (Taitt et al 2005). Optical systems do not interfere with metabolism, and thus in vivo measure- ments are possib e. In this chapter, some applications of the following optical sensing principles in foodborne pathogen detection are discussed: (1) SPR, (2 resonance mirror (RM), (3) fiber-optic biosensor, (4) array biosensor, (5) Ramar spectroscopy, and (6) light-addressable potentiometric sensor (LAPS) (Knopk et al. 2007c).
Fig. 6 Standard curve for determination of Aflatoxin B1 using biosensor AvaSpec — Ox} Fig. 5 Stability of microbial lipoxygenase immobilized on granulated polymer carrier in time In a measurement cell 20 mL of 0.1 Tris-buffer with pH 7.2 the respective sensor was incubated for 3 min with aflatoxin B1 in concentration range of 0.0 5—0.5 uM oreviously diluted in dimethyl sulphoxide. After 3 min of incubation at 37°C 0.1 mL linoleic acid was added to the samples so that the final concentration of the sample was 4 uM. Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig. 6).
Fig. 6 Standard curve for determination of Aflatoxin B1 using biosensor AvaSpec — Ox} Fig. 5 Stability of microbial lipoxygenase immobilized on granulated polymer carrier in time In a measurement cell 20 mL of 0.1 Tris-buffer with pH 7.2 the respective sensor was incubated for 3 min with aflatoxin B1 in concentration range of 0.0 5—0.5 uM oreviously diluted in dimethyl sulphoxide. After 3 min of incubation at 37°C 0.1 mL linoleic acid was added to the samples so that the final concentration of the sample was 4 uM. Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig. 6).
Table 2 Statistical processing of standard curves The obtained standard curves were statistically processed and are shown in Table 2. above. The nerformed investiscations nrove that they can he ncsed for meagurine real food.
Table 2 Statistical processing of standard curves The obtained standard curves were statistically processed and are shown in Table 2. above. The nerformed investiscations nrove that they can he ncsed for meagurine real food.
Table 3 Investigation on the activity of the free and immobilized enzyme
Table 3 Investigation on the activity of the free and immobilized enzyme
Fig. 8 Effect of pH on the activity of soluble and immobilized peroxidase
Fig. 8 Effect of pH on the activity of soluble and immobilized peroxidase
Fig. 9 Effect of temperature on soluble and immobilized peroxide
Fig. 9 Effect of temperature on soluble and immobilized peroxide
Table 4 Kinetic parameters of soluble and immobilized peroxidase Acknowledgment This review is supported for publication from NATO Scientific Committee and our research investigation was financial supported by project 322, National Fund “Scientific Investigations” of Republic of Bulgaria.
Table 4 Kinetic parameters of soluble and immobilized peroxidase Acknowledgment This review is supported for publication from NATO Scientific Committee and our research investigation was financial supported by project 322, National Fund “Scientific Investigations” of Republic of Bulgaria.
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