Bovine Spongiform Encephalopathy - A Review from the Perspective of Food Safety.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease that belongs to transmissible spongiform encephalopathy (TSE). Since the first case was identified in the UK in 1986, BSE spread to other countries including Japan. Its incidence peaked in 1992 in the UK and from 2001 to 2006 in many other countries, but a feed ban aimed at eliminating the recycling of the BSE agent and other control measures aimed at preventing food and feed contamination with the agent were highly effective at reducing the spread of BSE. In 2004, two types of atypical BSE, H-type BSE (H-BSE) and L-type BSE (L-BSE), which differ from classical BSE (C-BSE), were found in France and Italy. Atypical BSE, which is assumed to occur spontaneously, has also been detected among cattle in other countries including Japan. The BSE agent including atypical BSE agent is a unique food-safety hazard with different chemical and biological properties from the microbial pathogens and toxic chemicals that contaminate food. In this review, we summarize the reported findings on the tissue distribution of BSE prions in infected cattle and other aspects of BSE, as well as the control measures against the disease employed in Japan. Topics that require further studies are discussed based on the summarized findings from the perspective of food safety.

Occurrence and Health Risk of Patulin and Pyrethroids in Fruit Juices Consumed in Bangkok, Thailand.

The mycotoxin patulin (PAT) is well known as a natural contaminant of apple- and other fruit-based products. Pesticides are a group of chemicals abundantly used in agriculture to maximize productivity by protecting crops from pests and weeds. Because of their harmful health effects, PAT and pesticides are strictly monitored. The current study was undertaken to investigate the significance of PAT and pyrethroid insecticide contamination in a variety of fruit juices in Bangkok. To do this, a total of 200 fruit juice samples, consisting of 40 samples each of apple, apricot, peach, pineapple, and grape juice, were collected from supermarkets in Bangkok, Thailand. PAT contamination in a variety of fruit juices was detected using validated liquid chromatography-tandem mass spectrometry, and pyrethroid insecticides (cypermethrin, cyfluthrin, and flumethrin) were analyzed using a gas chromatography equipped with micro-electron capture detector. The survey found that PAT concentrations were lower than the maximum residue limit established by European Union. The results of the present study suggest that the risk of exposure to harmful levels of PAT, cypermethrin, cyfluthrin, and flumethrin in fruit juices is very low in urban areas of Thailand.

Inhibitory Effects of Thai Essential Oils on Potentially Aflatoxigenic Aspergillus parasiticus and Aspergillus flavus.

　The antiaflatoxigenic and antifungal activities of essential oils (EOs) of finger root (Boesenbergia rotunda (L.) Mansf.), pine (Pinus pinaster), rosewood (Aniba rosaedora), Siam benzoin (Styrax tonkinensis), Thai moringa (Moringa oleifera), and ylang ylang (Cananga odorata) were tested for Aspergillus parasiticus and Aspergillus flavus in potato dextrose broth. Aflatoxin B1 (AFB1) was extracted from culture using a QuEChERS-based extraction procedure and analyzed with high performance liquid chromatography (HPLC) coupled to a fluorescence detector. EO of pine showed the greatest inhibition of growth and AFB1 production of A. parasiticus, followed by EOs of rosewood, finger root, Siam benzoin, and ylang ylang. EO of finger root gave the best inhibitory effects on A. flavus, followed by EOs of rosewood, pine, ylang ylang, and Siam benzoin. EO of Thai moringa did not show any significant inhibition of aflatoxigenic fungi. The antiaflatoxigenic activities of EOs correlated with their antifungal activities in the dosedependent manner. Comparison of the application of the five selected EOs in peanut pods by direct and vapor exposure indicated that the AFB1 production inhibitory effects of the five EOs by direct exposure were faster and more effective than by vapor exposure. EO of finger root showed the best inhibition of AFB1 production of A. flavus in peanut pods by direct exposure, followed by EOs of pine, rosewood, ylang ylang, and Siam benzoin.

Possible Role of Phosphatidylcholine and Sphingomyelin on Fumonisin B1-mediated Toxicity.

A major corn-related mycotoxin, fumonisin B1 (FB1), continues to attract attention of researchers as well as risk-assessors due to the diverse toxicological characteristics, including distinct target tissues in different animal species and opposite susceptibility in males and females in mice and rats. More than thirty years passed since the structure identification as a sphingoid-like chemical, but the causal mechanism of the toxicity remains obscure in spites of extensive studies. Considerable amounts of knowledge have been accumulated on the biochemical/toxicological actions of FB1, but the influence on lipid dynamics and mobilization in the body has not been focused well in relation to the FB1-mediated toxicity. Considerable influences of this toxin on mobilization of sphingolipids and phospholipids and also on adaptive changes in their compositions in tissues are implicated from recent studies on FB1-interacting ceramide synthases. Accumulated patho-physiological data also suggest a possible role of hepatic phospholipid on FB1-mediated toxicity. Thus, a mechanism of FB1-mediated toxicity is discussed in relation to the mobilization of phospholipids and sphingolipids in the body in this context.

Survey of Deoxynivalenol and Aflatoxin B1 in Instant Noodles and Bread Consumed in Thailand by Using Liquid Chromatography-Tandem Mass Spectrometry.

One hundred wheat product samples (50 instant noodle samples and 50 bread samples) were collected from supermarkets in Bangkok, Thailand. Deoxynivalenol (DON) and aflatoxin B1 (AFB1) contamination in these products was analyzed using a validated liquid chromatography-tandem mass spectrometry method. The limit of quantification values of DON and AFB1 in the instant noodles and bread were 2 and 1 ng g(-1), respectively. The survey found that DON was quantifiable in 40% of collected samples, in 2% of noodles (0.089 µg g(-1)), and in 78% of breads (0.004 to 0.331 µg g(-1)). AFB1 was below the limit of quantification of the method in all of the tested samples. The results suggest that the risk of DON exposure via noodles and breads is very low in urban areas of Thailand. No risk can be attributable to AFB1 exposure in the same food matrices, but further studies with a larger sample size are needed to confirm these data.

Simultaneous detection of multiple mycotoxins in broiler feeds using a liquid chromatography tandem-mass spectrometry.

Mycotoxins are secondary fungal metabolites that are typically present in grain and feed ingredients used for animal feeds. An analytical method using LC-ESI-MS/MS was developed to quantify nine mycotoxins, consisting of aflatoxin B1 (AFB1), AFB2, AFG1, AFG2, T-2 toxin, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and ochratoxin A (OTA) in broiler feeds. In total, 100 samples of broiler feeds were collected from poultry farms in Central Thailand. The survey found that AFB1 and ZEA were the most prevalent mycotoxins in the feed samples at percentages of 93% and 63%, respectively. The limit of detections (LODs) of investigated mycotoxins was 0.20-0.78 ng/g. AFB2, DON, AFG1, NIV and T-2 toxin were also detectable at low contamination levels with percentages of 20%, 9%, 7%, 5% and 1%, respectively, whereas OTA and AFG2 were not detected in any of the feed samples. These results suggest that there is a very low level of risk of the exposure to mycotoxins in feeds obtained from broiler farms in Central Thailand.

Dispositions of enrofloxacin and its major metabolite ciprofloxacin in Thai swamp buffaloes.

Given the limited information available in this species, the aim of this study was to investigate the pharmacokinetic characteristics of enrofloxacin (ER) and its major metabolite ciprofloxacin (CP) in buffaloes, Bubalus bubalis. ER was administered intravenously (i.v.) or subcutaneously (s.c.) to buffaloes at doses of 5.0 and 7.5 mg/kg BW, and plasma, urine and fecal samples were collected until 48 hr post-administration. The concentrations of ER and CP in the plasma, urine and feces were analyzed using high-performance liquid chromatography equipped with a fluorescence detector. The plasma concentrations of ER and CP could be determined up to 24 hr and 32 hr after i.v. and s.c. administrations at doses of 5.0 and 7.5 mg/kg BW, respectively. CP concentrations were always lower than those of parental drug. The s.c. bioavailability of ER was 52.36 ± 4.24% and 72.12 ± 5.39% at doses of 5.0 and 7.5 mg/kg BW, respectively. Both ER and CP were detectable in urine and feces up to 24 hr. ER and CP were mainly excreted via the urine. Based on the pharmacokinetic data and PK-PD indices, s.c. administration of ER at doses of 5.0 and 7.5 mg/kg BW might be appropriate for the treatment of susceptible bacterial diseases in Thai swamp buffaloes.

The Effects of Curcumin on Aflatoxin B1- Induced Toxicity in Rats.

To evaluate the potential of curcumin on toxic and carcinogenic effects of Aflatoxin B1 (AFB1) in relation to AFB1 metabolism, we studied the effects of curcumin on hepatic AFB1-DNA adduct formation and glutathione S-transferase (GST) activity, and the toxic effects of AFB1 in male Fischer 344 rats. Oral administration of curcumin to 5-week-old male rats at a dose of 8 or 80 mg/kg for five consecutive days for three weeks resulted in reduction of AFB1-DNA adduct formation mediated by both liver microsomal and postmitochondrial fractions. The activity of liver GST toward a universal substrate, CDNB, was increased in curcumin-administered rats. As for the acute toxicity of AFB1, curcumin was orally administered to rats for 3 weeks and then AFB1 was given by intragastric intubation. The result showed a decrease of plasma AST and ALT activities in curcumin-treated rats compared with those which received AFB1 alone. Moreover, we have observed that curcumin also reduced glutathione S-transferase placental form positive single cells and foci caused by AFB1 treatment. These results demonstrate the potential of curcumin to reduce the toxic and carcinogenic effects of AFB1 by modulating hepatic drug metabolizing enzymes responsible for AFB1 metabolism.

The fate and tissue disposition of deoxynivalenol in broiler chickens.

To evaluate the fate of deoxynivalenol (DON) in broilers, DON was administered either intravenously or orally to broilers at a dose of 1 mg/kg BW. Concentrations of DON in plasma were measurable up to 4 hr and 2 hr after intravenous and oral administration, respectively. Following intravenous administration, the values for the elimination half-life, the volume of distribution and the clearance were 1.25 ± 0.25 hr, 7.55 ± 2.03 l/kg and 4.16 ± 0.42 l/hr/kg, respectively. The oral bioavailability was 15.46 ± 4.02%. DON was detectable in all tissues examined after oral administration. These results suggest that DON is able to penetrate into the various tissues in broilers, though poorly absorbed from their gastrointestinal tract.

Apoptosis and gene expression in the developing mouse brain of fusarenon-X-treated pregnant mice.

Fusarenon-X (FX), a type B trichothecene mycotoxin, is mainly produced by Fusarium crookwellense, which occurs naturally in agricultural commodities, such as wheat and barley. FX has been shown to exert a variety of toxic effects on multiple targets in vitro. However, the embryonic toxicity of FX in vivo remains unclear. In the present study, we investigated FX-induced apoptosis and the relationship between the genetic regulatory mechanisms and FX-induced apoptosis in the developing mouse brain of FX-treated pregnant mice. Pregnant mice were orally administered FX (3.5 mg/kg b.w.) and were assessed at 0, 12, 24 and 48 h after treatment (HAT). Apoptosis in the fetal brain was determined using hematoxylin and eosin staining, the TUNEL method, immunohistochemistry for PCNA and electron microscopy. Gene expressions were evaluated using microarray and real time-reverse transcription polymerase chain reaction (qRT-PCR). Histopathological changes showed that the number of apoptotic cells in the telencephalon of the mouse fetus peaked at 12 HAT and decreased at 24 and 48 HAT. FX induced the up-regulation of Bax, Trp53 and Casp9 and down-regulated Bcl2 but the expression levels of Fas and Casp8 mRNA remained unchanged. These data suggested that FX induces apoptosis in the developing mouse brain in FX-treated dams. Moreover, the genetic regulatory mechanisms of FX-induced apoptosis are regulated by Bax, Bcl2, Trp53 and Casp9 or can be defined via an intrinsic apoptotic pathway.

Use of blue-greenish yellow fluorescence test on feeds and its association with aflatoxin M1 contamination in bulk tank milk.

The objectives of this study were to use the blue-greenish yellow fluorescence (BY) test for aflatoxin M1 (AFM1) contamination in bulk milk and to examine the association between AFM1 contamination and environmental and feed management factors. The study was conducted March to May of 2011 with samples from 82 small holder dairy farms belonging to a single dairy cooperative in Chiang Mai province, Thailand. On the day of milk sample collection, samples of all feed used for milking cows and data on feed characteristics, feed management, and environmental factors also were collected at each farm. High-performance liquid chromatography was used to determine AFM1 concentrations in milk samples, and samples with AFM1 concentrations above the limit of detection were considered AFM1 contaminated. Fisher's exact tests were used to determine the association between AFM1 contamination in milk and farm management factors, feed management factors, and fungal contamination in feeds (as determined with the BY test). A multilevel logistic regression model was used to create the final model of factors associated with AFM1 contamination in milk. Feeds with fungal contamination (as determined by the BY test), high levels of cracked particles of commercial concentrate pellets, sunlight in the feed storage room, storage of commercial concentrates on the farm for more than 1 month, and more than 5% difference in relative humidity between the feed storage room and the barn holding lactating cows were associated with AFM1 contamination in milk. The BY test was useful for screening cattle feed for fungal contamination, and the results of this test in conjunction with other factors can be used to monitor and prevent AFM1 contamination in milk on small holder dairy farms.

Clinical pharmacokinetics and effects of vincristine sulfate in dogs with transmissible venereal tumor (TVT).

This study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (TVT). Dogs with TVT were intravenously administered vincristine sulfate at a dose of 0.7 mg/m(2) of body surface area. Blood samples were collected starting from 5 min to 48 hr after drug administration. The plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. The volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 ± 0.210 l/kg, 21.5 ± 6.90 min, 47.6 ± 14.2 min and 0.010 ± 0.001 l/min/kg, respectively. Tumor regression was determined at weekly interval by a physical examination and histopathological analysis. In our study, three to eight administrations of vincristine at a dose of 0.7 mg/m(2) were able to induce a complete tumor regression without any evidence of gross lesion of disease. Therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with TVT, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug's clinical effects. In addition, we validated the LC-MS/MS method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.

Spread and change in stress resistance of Shiga toxin-producing Escherichia coli†O157 on fungal colonies.

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.

Differences in the stress tolerances of Vibrio parahaemolyticus strains due to their source and harboring of virulence genes.

To investigate the diversity of stress tolerance levels in Vibrio parahaemolyticus, 200 V. parahaemolyticus strains isolated from various coastal environments, seafood, and human clinical cases were exposed to acid, low-osmolality, freezing-thawing, and heat stresses. Tolerance against acid stress was higher in the virulent (tdh- and/or trh-positive) strains than in the avirulent (tdh- and trh-negative) strains. Tolerance against low-osmolality, freezing-thawing, and heat stresses was higher in the clinical strains of tdh- and/or trh-positive V. parahaemolyticus than in the coastal environment- and seafood-originated strains of tdh and/or trh-positive V. parahaemolyticus. Tolerance against acid stress was higher in the strains isolated from coastal seawater at ≤15°C than in the strains isolated at ≥20°C. Tolerance against heat stress was higher in the avirulent strains than the virulent strains, and in the strains isolated from coastal seawater at ≥20°C than the strains isolated from coastal seawater at ≤15°C. Therefore, this study demonstrated that the diversity of stress tolerance levels in V. parahaemolyticus strains depended on their source and whether they harbored virulence genes. In particular, there was significantly greater tolerance against acid in the virulence gene-harboring strains and strains isolated from low-temperature seawater. Because the stress tolerances of V. parahaemolyticus have direct influences for the survival in environment and food, it is important for the prevention of foodborne infection to control the stress-tolerant strains.

Survival of Salmonella Enteritidis on four types of stainless steel surface under a dry condition and recovery by swabbing.

The survival and recovery of Salmonella Enteritidis inoculated on stainless steel surfaces with different metal contents and surface finishes were examined. Two S. Enteritidis strains possessing different levels of biofilm productivity were inoculated with tryptone soya broth (TSB) and egg yolk emulsion (EY) on the surface of stainless steel squares (1 cm × 1 cm) and stored at 22℃ under a dry condition. After storage, cells were recovered from the stainless steel surfaces by swabbing with a cotton swab. The numbers of cells recovered by swabbing and the cells remaining on the stainless steel squares were counted. The survival ratio of the strain possessing high biofilm productivity was greater than that of the strain possessing low biofilm productivity. The survival ratio of S. Enteritidis suspended in TSB was often higher than that in EY. There were no significant differences in the survival and recovery ratios of S. Enteritidis based on stainless steel composition or surface finish. From all except one sample, more than 98% of viable cells of S. Enteritidis were recovered by swabbing with a cotton swab.

Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity.

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

Decontamination of Vibrio parahaemolyticus in fish by washing with hygienic seawater and impacts of the high level contamination in the gills and viscera.

The effect of washing in Vibrio parahaemolyticus contaminated and hygienic seawater on fish, and the frequency and level of natural V. parahaemolyticus contamination in fish were investigated. In the first experiment, live horse mackerel was experimentally kept in seawater artificially contaminated with V. parahaemolyticus. After washing in contaminated and hygienic seawater, the contamination in fish was quantitatively analyzed. Washing fish in the seawater contaminated with V. parahaemolyticus increases the contamination level on the surface and in the gills of the fish. Washing in hygienic seawater was effective in reducing the contamination in fish and cutting board surfaces, but not in the gills or viscera. In the second experiment, natural V. parahaemolyticus contamination in various fish caught by us was analyzed. V. parahaemolyticus was detected in 6 of 28 gill samples and 10 of 28 viscera samples of naturally contaminated fish. The means of V. parahaemolyticus level on gills were 3.3 and 3.9 log cfu/g, and those in viscera were 2.6 and 4.4 log cfu/g by culture method and a real-time PCR assay, respectively. These results indicate that the gills and viscera are able to spread the pathogens to fish meat as well as fish surface contamination by washing in the contaminated seawater. Washing with hygienic seawater and control of contamination from gills and viscera are critically important to prevent V. parahaemolyticus infections.

Protective Effect of Stachybotrys microspora Triprenyl Phenol-7on the Deposition of IgA to the Glomerular Mesangium in Nivalenol-induced IgA Nephropathy Using BALB/c Mice.

Activators of tissue proteolysis including Stachybotrys microspora triprenyl phenol (SMTP)-7 are a new class of agents that are expected to be effective for amelioration of chronic tissue destructive diseases. The present study was performed to examine whether SMTP-7 is effective for the amelioration or protection of early-stage IgA nephropathy (IgAN) induced by nivalenol (NIV) in female BALB/c mice. In Experiment 1, mice were administered NIV at 24 ppm in diet for 8 weeks, and during the NIV treatment, they were intraperitoneally injected with SMTP-7 (10 mg/kg) three times a week. In Experiment 2, mice were injected similarly with SMTP-7 during the last 4 weeks of a 16-week NIV treatment. Immunofluorescence analysis revealed an inhibitory effect of SMTP-7 on the glomerular deposition of IgA in Experiment 1; however, it was ineffective in Experiment 2. On the other hand, SMTP-7 did not affect the serum concentration of IgA in both experiments. These results suggest that SMTP-7 has a potential to decrease the progression of IgAN induced by NIV through inhibition of local accumulation of IgA in the glomerular mesangium, while it was ineffective for suppression of IgA production. On the other hand, SMTP-7 was found to be ineffective for already deposited IgA, suggesting that SMTP-7 may not be effective for ameliorating advanced IgAN.

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan.

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.