Diarrheal Toxin Research Articles

A 2D carbon nitride-based electrochemical aptasensor with reverse amplification for highly sensitive detection of okadaic acid in shellfish.

Okadaic acid (OA) is one of the main virulence factors of diarrheal shellfish toxins (DSP). It is of great significance to detect OA with an accurate, specific and cost-effective technique in the fields of seafood safety and water quality control. In this work, an electrochemical aptasensor with reverse amplification was developed for the sensitive detection of OA. A two-dimensional graphite-phase nanomaterial (carbon nitride) modified with an anti-OA aptamer and thionine (Th) was immobilized onto the surface of the electrochemical electrode as the sensitive element to capture target OA molecules. ssDNA-modified carbon nitride was used as the reverse amplification element by hybridizing with non-OA linked aptamers. The preparation of the electrochemical aptasensor was well characterized by Scanning Electron Microscopy (SEM), zeta potential detection, UV-Vis absorption, Brunner-Emmet-Teller (BET) measurements, and electrochemical measurements. The quantitative assessment of OA was achieved by differential pulse voltammetry (DPV). Experimental results indicated that this aptasensor showed a concentration-dependent response to OA with a good detection performance including in terms of selectivity, repeatability, reproducibility, and stability. It exhibited 100-fold selectivity between OA and other toxins including dinophysistoxins (DTX), pectenotoxins (PTX), and yessotoxins (YTX). In addition, it showed a much wider quantification range, which is 10-13 M-10-10 M (0.080-80.50 pg mL-1). The detection limit was as low as 10-13 M (0.080 pg mL-1). The aptasensor also successfully achieved significant practicality on real shellfish samples contaminated by OA. All these results demonstrated that the reverse amplification strategy for marine toxin detection may provide a label-free and rapid detection approach for portable applications in the fields of environmental monitoring and food security.

Bacterial cellulose loaded with silver nanoparticles as a flexible, stable and sensitive SERS-active substrate for detection of the shellfish toxin DTX-1.

Diarrheal shellfish toxins are considered one of the most lethal red tide algae toxins in the worldwide. In this work, we propose an Ag NPs-loaded bacterial cellulose membrane (BCM) surface-enhanced Raman scattering (SERS) sensor based on an aptamer (Apt) for the ultrasensitive detection of dinophysistoxin (DTX-1), a type of diarrheal shellfish toxin. During drying, Ag NPs can be further densified on "gel-like" BCM to form high-density SERS "hot spots". We developed the "Apt-SH@Ag NPs@BCM" SERS sensor and used the competition of DTX-1 and complementary base (Cob) in the process of base complementary pairing to achieve SERS detection of DTX-1, with a minimum detection limit of 9.5×10-10mol/L. Sample assays showed DTX-1 recovery rates ranging from 95.8% and 108.2% and the detection results were comparable to those obtained by LC-MS. Therefore, this work holds great potential for detecting of toxic substances in shellfish products, especially for the oyster (portuguese oyster) and mussel (blue mussel).

Emetic and Diarrheal Toxins Detection and Typing of Bacillus cereus in Food and Human Milk in Italy

Abstract Background Bacillus cereus, responsible for 1.4%-12% of all food poisoning outbreaks worldwide, produces several virulence factors, including toxins which are mainly found in raw and processed food, as starch-based and dairy products. It can cause two types of gastrointestinal diseases: emetic form, with nausea and vomiting which appears as soon as after food consumption, and diarrheal form, with abdominal cramps, which occurs after 8-16 h. Moreover, B. cereus is the most common pathogen in pasteurized human milk, a precious aid in neonatal care. The aim of this study was to investigate the prevalence of B. cereus toxins kinds using WGS and to detect in silico sequence types (ST) in isolates from food and human milk. Methods During B. cereus routine surveillance in Lombardy (Italy), a total of 41 isolates were collected from food (n = 16) and donor breast milk (n = 25) between 2022-2023, and then analysed with WGS. Libraries were prepared using the Illumina DNA Prep Kit and WGS was performed on the Illumina MiSeq. Using the BTyper tool, for each sample emetic and diarrheal toxins were identified and ST was assigned. Results All 41 isolates had at least one diarrheal toxin, specifically the nonhemolytic enterotoxin was found in all the isolates, the cytotoxin K was detected in 37% of isolates, and the hemolysin BL in 32%. Instead, the emetic toxin was found in only 3 samples isolated from food. Using in silico MLST, different STs were found with a higher prevalence of ST26 and ST144. Conclusions Diarrheal toxins were detected in all samples underling that B. cereus food infections play an important role in the public health and in the food industry. These aspects are even more significant when the pathogen is isolated from human breast milk, as it can induce severe infections in newborns. Key messages • The presence of B. cereus toxins in food and human milk underlines the importance of its ongoing monitoring and analysis. • Human breast milk has a high risk of B. cereus contamination which is the most frequent cause for its discarding.

Exosomal miRNA analysis provides new insights into exposure to nanoplastics and okadaic acid

As an emerging environmental pollutant, nanoplastics (NPs) have attracted wide attention in terms of their impact on the ecological environment and human health. Currently, researches on the cytotoxicity of NPs mainly focus on oxidative stress, damage to the cell membrane and organelles, induction of immune response and genotoxicity. Okadaic acid (OA) is the main component of diarrheal shellfish toxin. Based on the previous combined toxicity exploration of polystyrene (PS) NPs and (OA) to human gastric adenocarcinoma (AGS) cells, cell-derived exosomes were extracted and exosomal miRNA profiles were analyzed for the first time in this study. The results showed that the composition of miRNAs varied after the exposure of NPs and OA. Specifically, the expression of miR-1-3p in both PS-Exo and PS-OA-Exo was significantly reduced. And the expression of miR-1248 was upregulated most significantly by comparing the DE miRNAs between PS-Exo and PS-OA-Exo. MiR-1-3p and miR-1248 may be the key genes for the combined toxicity of NPs and OA. After analysis, we found that both the decreased expression of miR-1-3p and the increased expression of miR-1248 can increase the expression of FN1 and affect DNA replication, which was surprisingly consistent with the results of our previous cytotoxicity studies. Since exosomal miRNAs are selectively encapsulated by donor cell, we speculate that the changes of exosomal miRNAs may due to the synchronous changes of intracellular environment and the downregulation of intracellular FN1 may be attributed to decreased expression of miR-1-3p and increased expression of miR-1248 in donor cells. Accordingly, we come to the conclusion that the changes of miRNAs in the exosomes derived from AGS cells after environmental stimulation could reflect the biological effects of donor cells.

Detection of diarrheal shellfish toxins

Abstract Seafood poisoning outbreaks are often caused by biotoxins generated by harmful algal blooms. Shellfish toxins, mainly derived from phytoplankton, cause diarrhea and poisoning in humans who consume contaminated seafood. Many studies suggest that diarrheal shellfish toxins cause functional changes in various cells. In order to protect shellfish products, it is becoming increasingly important to remove these contaminants from the ocean. Public attention plays a crucial role in reducing the risk of acute intoxication caused by contaminated seafood. Monitoring algal toxins is the best way to ensure that seafood is safe and clean. In order to attain these objectives, a variety of technologies were developed and constructed for the purpose of detecting and decontaminating algal toxins in aquatic environments. A review of the current literature regarding the detection and detoxification of diarrheal shellfish toxins is presented in this article.

Genetic diversity of enterotoxigenic Bacillus cereus strains in coriander in southwestern Mexico.

BackgroundCoriander, like other leafy green vegetables, is available all year round and is commonly consumed raw in Mexico as in other countries in the preparation of street or homemade food. Bacillus cereus (B. cereus) is a microorganism that can reach coriander because it is usually found in the soil and in some regions the vegetables are irrigated with polluted water. Therefore, the aim of this study was to determinate the presence of B. cereus in coriander used for human consumption in southwestern Mexico and determine the toxigenic profile, biofilm production, genes associated with the production of biofilms, sporulation rates, enzymatic profile, psychotropic properties, and genetic diversity of B. cereus.MethodsFresh coriander samples were collected from several vegetable retailers in different markets, microbiological analysis was performed. Molecular identification, genes related to the production of biofilm, and toxin gene profiling of B. cereus isolates were determined by PCR. The biofilm formation was measured by performing a crystal violet assay. The genetic diversity of B. cereus strains was determined by PCR of repetitive elements using oligonucleotide (GTG) 5.ResultsWe found a frequency of B. cereus in vegetables was 20% (13/65). In this study, no strains with genes for the HBL toxin were found. In the case of genes related to biofilms, the frequency was low for sipW [5.8%, (1/17)] and tasA [11.7%, (2/17)]. B. cereus strains produce a low amount of biofilm with sporulation rates around 80%. As for genetic diversity, we observed that strains isolated from the same market, but different vegetable retailers are grouped into clusters. In the coriander marketed in southwestern Mexico, were found B. cereus strains with genes associated with the production of diarrheal toxins. Together, these results show actual information about the state of art of B. cereus strains circulating in the southwestern of Mexico.

A smartphone-assisted microarray immunosensor coupled with GO-based multi-stage signal amplification strategy for high-sensitivity detection of okadaic acid

Okadaic acid (OA) is one of the main virulence factors of diarrheal shellfish toxins (DSP), which can cause acute carcinogenic or teratogenic effects after ingestion of contaminated shellfish. Therefore, high-sensitivity and fast detection of OA is a key to preventing the occurrence of safety accidents. In this paper, we effectively established a smartphone-assisted microarray immunosensor combined with an indirect competitive ELISA (iELISA) for quantitative colorimetric detection of OA. To further improve the detection sensitivity and match the smartphone imaging, a novel graphene oxide (GO) composite probe was developed to realize the multi-stage signal amplification. The system exhibited a wide linear range for the detection of OA (0.02–33.6 ng ·mL−1) with low detection limit of 0.02 ng ·mL−1. The recovery of OA in spiked shellfish samples was in the range of 80%–103.5%, which indicates the good applicability of this biosensor. The whole detection system has advantages of simplicity, low cost, high sensitivity and portability, which is expected to be a powerful alternative tool for on-site detecting and early warning of the pollution of marine products.

Oral MucoRice-CTB vaccine is safe and immunogenic in healthy US adults

MucoRice-CTB is a promising cold-chain-free oral cholera vaccine candidate. Here, we report a double-blind, randomized, placebo-controlled, phase I study conducted in the USA in which vaccination with the 6-g dose of MucoRice-CTB induced cross-reactive antigen-specific antibodies against the B subunit of cholera toxin (CTB) and enterotoxigenic Escherichia coli heat-labile enterotoxin without inducing serious adverse events. This dosage was acceptably safe and tolerable in healthy men and women. In addition, it induced a CTB-specific IgA response in the saliva of two of the nine treated subjects; in one subject, the immunological kinetics of the salivary IgA were similar to those of the serum CTB-specific IgA. Antibodies from three of the five responders to the vaccine prevented CTB from binding its GM1 ganglioside receptor. These results are consistent with those of the phase I study in Japan, suggesting that oral MucoRice-CTB induces neutralizing antibodies against diarrheal toxins regardless of ethnicity.

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms.

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

Detection of Bacillus cereus genes responsible for diarrheal and emetic toxins

Bacillus cereus isolated from different food sources. The diarrheal toxin genes such as cytK, hemolytic enterotoxin (hblA, hblC and hblD), non-hemolytic enterotoxin (nheA, nheB and nheC), bceT and entFM in addition to emetic toxin gene were detected by PCR. The cyt K gene was observed in 94.87 % of the isolates. entFM and emetic toxin gene were found very rare in all food samples at the percentage 2.56% and 7.69% respectively. Uncooked rice which has a highest number of bacterial isolation, also showed relatively high percentage of the cytK and bceT genes (90%). These two genes present in 100% of Bacillus cereus isolates in most food samples. Bacteria isolated from burger meat contain all investigated genes.

Risk of Bacillus cereus in Relation to Rice and Derivatives.

Rice is a very popular food throughout the world and the basis of the diet of the citizens of many countries. It is used as a raw material for the preparation of many complex dishes in which different ingredients are involved. Rice, as a consequence of their cultivation, harvesting, and handling, is often contaminated with spores of Bacillus cereus, a ubiquitous microorganism found mainly in the soil. B. cereus can multiply under temperature conditions as low as 4 °C in foods that contain rice and have been cooked or subjected to treatments that do not produce commercial sterility. B. cereus produces diarrhoeal or emetic foodborne toxin when the consumer eats food in which a sufficient number of cells have grown. These circumstances mean that every year many outbreaks of intoxication or intestinal problems related to this microorganism are reported. This work is a review from the perspective of risk assessment of the risk posed by B. cereus to the health of consumers and of some control measures that can be used to mitigate such a risk.

Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata).

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes—biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)—and two virulence factors—Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)—was investigated by RT-PCR. A. hydrophila displayed a > 2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a > 2-fold, and on polypropylene a > 6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

PCR assay a new approach for detection of enterotoxins

B. cereus group is considered as a potential problem, since it can contaminate many dairy products. In this study, three primer sets were selected to simultaneously detect two different species of the B. cereus group by using triple – primer PCR. The triple–primer PCR in this study were synthesized using the CER, CES and groEL genes for the detection of emetic toxin producing strains and another specific primer for the detection of diarrheal toxin (groEL gene only). Results indicated that all the diarrheal enterotoxin producing B. cereus strains showed a presence of groEL gene, while CER and CES genes were completely absent. Out of the six B. cereus strains tested for the production of diarrheal and emetic enterotoxins by using triple–primer PCR technique, three diarrheal enterotoxin producing strains were only detected. On the other hand, all six B. cereus strains had limited ability to produce emetic toxin. All diarrheal enterotoxin producing B. mycoides strains showed presence of groEL gene, but CER and CES genes were not detected in any of the B. mycoides tested strains. Consequently, four B. mycoides strains were tested for the production of diarrheal and emetic enterotoxins by using triple–primer PCR technique, only two strains showed diarrheal enterotoxin producing strains. In contrary all B. mycoides tested strains had limited ability to produce emetic toxin.

Needle- and Cold Chain- Free Rice-Based Oral Vaccine, MucoRice-CTB Shows Microbiota-Dependent Immunogenicity in Humans

Background: Diarrheal disease remains a global health issue. It is estimated that there are 1.3 to 4.0 million cases of cholera and 20,000 to 140,000 cholera-related deaths worldwide each year. In addition, pandemic diarrhea and travelers’ diarrhea caused by enterotoxigenic Escherichia coli heat-labile enterotoxin (LT) are estimated to affect approximately 200 million and 27 million people, respectively, worldwide each year. Our previous studies showed that rice-based cholera toxin B subunit (CTB) vaccine, MucoRice-CTB is a cold-chain and needle-syringe free oral vaccine candidate which will be benefited to less infrastructure conditions. Methods: We conducted a double-blind, randomized, placebo-controlled, three-cohort, dose-escalation phase I study to evaluate the safety, tolerability, and efficacy of MucoRice-CTB (clinical study code name: IMSUT-MR1501) administered as four oral doses at 2-week intervals to healthy Japanese adults. In addition to vaccine evaluation, metagenomic analysis of study participants was performed by using bacterial DNA from fecal samples. Interpretation: MucoRice-CTB induces cross-reactive antigen-specific antibodies against CTB and B subunit of LT in a dose-dependent manner, without inducing serious adverse events. Antibodies isolated from responders to the vaccine inhibited the binding of CT and LT to GM1 ganglioside receptor and the elongation of Chinese hamster ovary cells caused by the both enterotoxins. Metagenome analysis of subjects’ feces collected prior to the start of vaccination revealed that responders had a gut microbiota of higher diversity that was characterized by larger populations of E. coli and Shigella spp. compared with those in non-responders. Findings: This phase I clinical trial showed that MucoRice-CTB induces neutralizing antibodies against diarrheal toxins in a gut microbiota-dependent manner without major adverse events. Trial Registration: (clinical trial registration: UMI18001) Funding Statement: The costs related to this study are covered by support of the “Translational Research Network Program” of the Ministry of Education, Culture, Sports, Science and Technology. Declaration of Interests: The authors stated that: None of the items involve any conflicts of interest. Ethics Approval Statement: The clinical research protocols were approved and received ethical clearance (permit #27-41- 0917 and #27-48-0128) from the IRB of the Institute of Medical Science, The University of Tokyo (IMSUT), Japan.

Presence of nhe, cytK, and ces in Bacillus cereus isolated from dairy products commercially available in Brazil

AbstractThe aim of this study was to evaluate the presence of genes (nheABC, cytK, and ces) among bacteria present in dairy products (100 pasteurized milk, 110 UHT milk, and 50 powdered milk). In total, 63 Bacillus cereus isolates were tested and subjected to the detection of genes by using PCR. Regardless of the type of sample analyzed, the most frequent were nheC (82.5%), cytK (68.2%), and nheA (66.6%). The genes nheB and ces were detected in only 23.8% and 3.1% of the isolates, respectively. None of the isolates presented the three genes simultaneously. The combination of nheABC‐cytK was highly prevalent in most of the isolates, present in 25% from pasteurized milk and 20% of those from powdered milk. None of the cultures showed nheABC and ces simultaneously, and the combination of cytK‐ces was found in only one isolate from pasteurized milk (2.7%) and one isolate in powdered milk (6.6%). These results demonstrate the presence of B. cereus toxin genes in dairy products from Brazil.Practical applicationsConsumption of food contaminated with Bacillus cereus is a risk to public health as this micro‐organism produces diarrheal and emetics toxins. Thus, studies that aim to research genes that code for these toxins can be important tools for dairy industries to establishment of process parameters, limits and actions to control of contamination. This article demonstrated that strains of B. cereus isolated from milk and dairy products may contain genes encoding the toxins (nhe, cytK, and ces), so the risk of food poisoning should not be neglected.

Toxigenic and pathogenic potential of enteric bacterial pathogens prevalent in the traditional fermented foods marketed in the Northeast region of India

The microbial risk involved with natural food fermentation is largely unknown. Here, we report the prevalence of enteric bacterial pathogens in the traditional fermented foods marketed in Northeast region of India. A total of 682 samples of 39 food types (broadly categorized into fermented soybean, bamboo shoot, fish, milk and pork products) collected over four different seasons from seven states of India were analyzed in this study. Cultivation-independent analysis by MiSeq amplicon sequencing of V4-V5 region of the 16S rRNA gene showed the bacterial community structure in the foods. Among the WHO prioritized foodborne bacterial pathogens, we detected the prevalence of phylotypes related to Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, and Escherichia coli in these ethnic foods. We also observed the occurrence of other well known human enteric pathogens like Proteus mirabilis, Clostridium difficile, and Yersinia enterocolitica. Further pathogen-specific qPCR assays confirmed a higher population (>107 cells/g) of B. cereus, P. mirabilis, and a C. botulinum related phylotype in the fermented soybean, fish, and pork products. We noticed a general trend of higher pathogen occurrence during the colder months without any seasonal variation of total bacterial load in the fermented foods. Further qPCR analysis on toxigenic and pathogenic potential, and toxins production by immunoassays showed that all the soybean samples and the isolated B. cereus cultures were positive for diarrheal toxins (Nhe and Hb1), and nearly half of the samples were positive for emetic toxin (cereulide). Similarly, the food samples and associated swarming P. mirabilis cultures were positive with the pathogenic factors like hemolysin (hpm), urease (ure) and multidrug resistance. However, we could not confirm the presence of botulinum neurotoxin (toxins A, B, E, and F) in the C. botulinum positive food samples. This is the first baseline data of the enteric bacterial pathogens prevalent in the traditional fermented foods of India, which will support the sustained effort of WHO to estimate the global foodborne disease burden. The unusual presence of P. mirabilis in the fermented foods marketed in the Indian region with high incidence of urolithiasis cases is a concern. Our study emphasizes the need of the hour to have a coordinated action to control and prevent the spread of enteric bacterial pathogens through fermented foods marketed in India. Moreover, replacing the indigenous process with a defined starter culture based controlled fermentation will enhance the safety of Indian fermented foods.

Isolation of Bacillus cereus in school restaurants in Colombia

Introduction: Bacillus cereus is recognized as a pathogen that causes food poisoning. It is a facultative aerobic metabolism bacterium capable of forming spores, which allows it to survive pasteurization and heating even by the gamma irradiation used to reduce pathogens in food. Objective: To study the presence of Bacillus cereus and its diarrheal toxin in rice and ready-to-eat cereals, flours, and starches in school restaurants in Colombia. Materials and methods: We conducted a descriptive cross-sectional study of ready-to-eat foods distributed in school restaurants in the departments with the most and the least notification of foodborne diseases to the surveillance system. Results: A total of 479 samples were collected from eight departments, 74 municipalities, and 363 school restaurants, 63% of which were rice samples and 37%, starchy food samples; 9% of them tested positive for Bacillus cereus. In 91% of the samples that tested positive, the bacterium was isolated with the presence of the diarrheal toxin. Conclusions: In all the departments with B. cereus in the samples, the factors directly related to food-borne diseases were the handling of raw materials and the poor thermal treatment of food. Strengthening surveillance by stimulating research and reporting on outbreaks of foodborne diseases is important to improve the quality of information, to develop communication, prevention and intersectional coordination and manipulation measures, as well as to take the necessary actions to guarantee the safety of food and to eliminate the risk factors that may contribute to this problem.

A quantitative microbiological exposure assessment of Bacillus cereus group IV in couscous semolina, Algeria

Couscous is widely consumed in Algeria, but it has been associated with several outbreaks. Couscous semolina may contain heat-resistant spores of Bacillus cereus group IV. These bacteria are mesophilic and potential producers of diarrheal toxin. The present study aimed at predicting (i) the prevalence and concentration of B. cereus group IV during couscous preparation at the time of consumption, and (ii) the number of consumers exposed to 105 cfu/g or more B. cereus in Algeria, due to couscous consumed at home or during collective events. In a probabilistic exposure assessment model, couscous semolina preparation was divided into three modules (i) contamination at retail, (ii) inactivation of B. cereus during couscous steaming and (iii) growth during storage before serving. Each module was built using a combination of newly gathered data, literature studies, predictive models and expert opinions. Algeria was split into four regions: North, South, High Plateau and Kabylia. The main differences between the regions were the seasonal temperature and the consumption pattern. Annually, a total of 3000 people (0.007% total population) and 170 people (0.0004%) were estimated to be exposed to a concentration equal to or above 105 cfu/g, due to home and collective consumption, respectively. The highest exposure came from couscous semolina kept at ambient temperature, in the south of Algeria during summer. Cold storage and consumption within the first 12 h after steaming are factors which limit the exposure to B. cereus group IV in couscous.

Yessotoxin: risk assessment for public health. Justification of regulations of content in seafood

Yessotoxin and its derivatives (about 90) are isolated from algae belonging to the species Protoceratium reticulatum, Gonyaulax cf. Spinifera, Lingulodinium polyedrum and from invertebrate organisms that feed on these algae. Previously yessotoxin have been associated with the group of diarrheal toxins. Later studies of the possible impact of yessotoxin on the activity of alkaline phosphatase allowed to exclude them from this group. Yessotoxin causes a violation of calcium entry in the cells, which, in turn, effects the calcium-calmodulin system and thus influences into homeostasis of the organism as a whole. It was shown that yessotoxin induces a biphasic change in the concentration of adenosine monophosphate, an initial increase with a subsequent relative decrease, within some minutes after adding the toxin to the lymphocytes cell culture. Yessotoxin has effects on immune system; which is manifested in an increase of cytokines level, by inducing the expression of the genes encoding them. Yessotoxin have impact into processes of cell adhesion via E-cadherin and, thus, could be an important factor in the development of Alzheimer's disease. It has been established that yessotoxin caused the development of apoptosis. In those cases all three mechanisms of cell death took place - apoptosis, paraptosis and autophagy. Yessotoxin's acute toxicity doses according to different data are from 100 to 500-750 μg per 1kg of body weight. Yessotoxin's acute reference dose (ARfD) - 25 μg/kg of body weight per day. The results of the analysis of yessotoxin level in shellfish meat showed that none of the studied samples contained more than 3.75 mg yessotoxin equivalents/kg shellfish meat. This level has been adopted by the European Union as the maximum acceptable level of yessotoxin in shellfish meat (EU Regulation N 786/2013). Presented data on the mechanism of action, toxicity and prevalence of yessotoxins make it necessary to establish regulations of their content in seafood, placed on the markets of the Eurasian Economic Union.

Effects of temperature, pH and curing on the viability of Sarcocystis, a Japanese sika deer (Cervus Nippon centralis) parasite, and the inactivation of their diarrheal toxin.

Recently, the Sarcocystis parasite in horse and deer meat has been reported to be a causative agent of acute food poisoning, inducing nausea, vomiting and diarrhea. Compared with other causative agents, such as bacteria, viruses and other parasites, in deer meat, the Sarcocystis species parasite, including its stability under various conditions, is poorly understood. In this study, we assessed the viability of Sarcocystis spp. and the activity of their diarrhea toxin (a 15-kDa protein) in deer meat under conditions of freezing, cold storage, pH change and curing. In addition, the heat tolerance was assayed using purified bradyzoites. The results showed that the species lost viability by freezing at −20, −30 and −80°C for <1 hr, heating at 70°C for 1 min, alkaline treatment (pH 10.0) for 4 days and addition of salt at 2.0% for <1 day. Immunoblot assays showed that the diarrhea toxin disappeared together with the loss of viability. However, the parasite survived cooling at 0 and 4°C and acidification (pH 3.0 and 5.0) for more than 7 days with the diarrhea toxin intact. These results provide useful information for developing practical applications for the prevention of food poisoning induced by diarrheal toxin of Sarcocystis spp. in deer meat during cooking and preservation.