Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters.

The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. Understanding the toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events.

The Southeast Alaska Tribal Ocean Research (SEATOR) Partnership: Addressing Data Gaps in Harmful Algal Bloom Monitoring and Shellfish Safety in Southeast Alaska.

Many communities in Southeast Alaska harvest shellfish such as mussels and clams as an important part of a subsistence or traditional diet. Harmful algal blooms (HABs) of phytoplankton such as Alexandrium spp. produce toxins that can accumulate in shellfish tissues to concentrations that can pose a hazard for human health. Since 2013, several tribal governments and communities have pooled resources to form the Southeast Alaska Tribal Ocean Research (SEATOR) network, with the goal of minimizing risks to seafood harvest and enhancing food security. SEATOR monitors toxin concentrations in shellfish and collects and consolidates data on environmental variables that may be important predictors of toxin levels such as sea surface temperature and salinity. Data from SEATOR are publicly available and are encouraged to be used for the development and testing of predictive algorithms that could improve seafood risk assessment in Southeast Alaska. To date, more than 1700 shellfish samples have been analyzed for paralytic shellfish toxins (PSTs) in more than 20 locations, with potentially lethal concentrations observed in blue mussels (Mytilus trossulus) and butter clams (Saxidomus gigantea). Concentrations of PSTs exhibit seasonality in some species, and observations of Alexandrium are correlated to sea surface temperature and salinity; however, concentrations above the threshold of concern have been found in all months, and substantial variation in concentrations of PSTs remain unexplained.

Paralytic Shellfish Toxins (PST)-Transforming Enzymes: A Review.

Paralytic shellfish toxins (PSTs) are a group of toxins that cause paralytic shellfish poisoning through blockage of voltage-gated sodium channels. PSTs are produced by prokaryotic freshwater cyanobacteria and eukaryotic marine dinoflagellates. Proliferation of toxic algae species can lead to harmful algal blooms, during which seafood accumulate high levels of PSTs, posing a health threat to consumers. The existence of PST-transforming enzymes was first remarked due to the divergence of PST profiles and concentrations between contaminated bivalves and toxigenic organisms. Later, several enzymes involved in PST transformation, synthesis and elimination have been identified. The knowledge of PST-transforming enzymes is necessary for understanding the processes of toxin accumulation and depuration in mollusk bivalves. Furthermore, PST-transforming enzymes facilitate the obtainment of pure analogues of toxins as in natural sources they are present in a mixture. Pure compounds are of interest for the development of drug candidates and as analytical reference materials. PST-transforming enzymes can also be employed for the development of analytical tools for toxin detection. This review summarizes the PST-transforming enzymes identified so far in living organisms from bacteria to humans, with special emphasis on bivalves, cyanobacteria and dinoflagellates, and discusses enzymes' biological functions and potential practical applications.

First detection of benthic cyanobacteria in Lake Baikal producing paralytic shellfish toxins.

Cyanobacteria were screened from the surface of diseased sponges, stone and bedrock in Lake Baikal for the presence of saxitoxin using enzyme-linked immunosorbent assay. In sequel, eight paralytic shellfish toxin (PST) variants were identified using a MALDI mass spectrometry. Microscopic examination found that Tolypothrix distorta dominated in the biofouling samples. PCR and sequencing detected sxtA gene involved in saxitoxin biosynthesis, thereby providing evidence of the PST producing potential of Baikal cyanobacterial communities inhabiting different substrates.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n=256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2×2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

An outbreak of gastroenteritis linked to a buffet lunch served at a Canberra restaurant.

In 2013, an outbreak of gastrointestinal illness occurred following a buffet lunch at a restaurant in Canberra. An investigation was conducted to identify the cause of illness and to implement appropriate public health measures to prevent further disease. We conducted a retrospective cohort study via telephone interviews, using a structured questionnaire developed from the restaurant buffet menu. A case was defined as someone who ate the buffet lunch at the restaurant on the implicated date and developed any symptoms of gastrointestinal illness (such as diarrhoea, abdominal pain and nausea) following the consumption of food. A total of 74% (225/303) of known attendees were interviewed, of whom 56% (125/225) had become ill. The median incubation period and duration of illness were 13 and 19 hours respectively. The most commonly reported symptoms were diarrhoea (94%, 118/125) and abdominal pain (82%, 103/125). A toxin-mediated gastrointestinal illness was suspected based on the incubation period, duration of illness and the symptoms. The environmental health investigation identified a lack of designated hand washing facilities in the kitchen, an absence of thermometers for measuring food temperatures and several maintenance and minor cleaning issues. A number of food samples were taken for microbiological analysis. Multivariable analysis showed that illness was significantly associated with consuming curried prawns (OR 18.4, 95% CI 8.6-39.3, P < 0.001) and Caesar salad (OR 3.6, 95% CI 1.8-7.5, P 0.001). Enterotoxin-producing Staphylococcus aureus and Bacillus cereus were identified in leftover samples of cooked buffet food, but this food was not epidemiologically implicated. The investigation suggested that a breakdown in cleanliness, temperature control and food handling practices may have resulted in contamination of the buffet food. In order to prevent such outbreaks in the future, caterers and restaurateurs need to ensure they have the appropriate facilities and procedures in place if planning to cater for large groups.

Molecular characterization of Listeria monocytogenes isolated from fresh seafood samples in Iran.

BACKGROUND: Among all species of Listeria, Listeria monocytogenes (L. monocytogenes) is a major pathogenic microorganism of humans and animals and L. ivanovii is rarely pathogenic for humans. The objective of this study was to isolate and characterize Listeria species and to determine the frequencies of virulence genes in L. monocytogenes serotypes in fresh fish, shrimp, crab and lobster in Isfahan and Shahrekord, Iran. METHODS: From September 2010 to April 2011, a total of 300 marine food samples were purchased from supermarkets of Isfahan and Shahrekord cities, Iran. All samples were cultured and the positive samples for L. monocytogenes were analyzed for presence of serotypes and virulence genes. RESULTS: From the total 300 samples, 23 (10.45%) fresh fish and 1 (2.5%) shrimp samples were positive for Listeria spp., but there were no positive lobster and crab samples for Listeria species. Only L. monocytogenes was isolated from 17 fish (7.25%) and 1 shrimp (2.5%) samples while L. innocua, L. ivanovii and L. seeligeri only detected in fish samples (2 (0.9%), 3 (1.36%) and 1 (0.45%)), respectively. The plcA, prfA, actA, hlyA and iap virulence genes were detected in all of the 18 L. monocytogenes isolates. Totally, the 4b, 1/2a and 1/2b serotypes were detected in 66.66%, 5.55% and 27.77% bacterial isolates, respectively. CONCLUSIONS: Consumption of these sea foods, either raw or undercooked, may contribute to food-borne illness due to L. monocytogenes in Iran. The hygienic quality of sea food products should be observe. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3422944359800606.

The interactions of Vibrio vulnificus and the oyster Crassostrea virginica.

The human bacterial pathogen, Vibrio vulnificus, is found in brackish waters and is concentrated by filter-feeding molluscan shellfish, especially oysters, which inhabit those waters. Ingestion of raw or undercooked oysters containing virulent strains of V. vulnificus can result in rapid septicemia and death in 50 % of victims. This review summarizes the current knowledge of the environmental interactions between these two organisms, including the effects of salinity and temperature on colonization, uptake, and depuration rates of various phenotypes and genotypes of the bacterium, and host-microbe immunological interactions.

Vibrio vulnificus: death on the half shell. A personal journey with the pathogen and its ecology.

Vibrio vulnificus is an estuarine bacterium which occurs in high numbers in filter-feeding molluscan shellfish, such as oysters. In individuals with certain underlying diseases, ingestion of the bacterium, e.g., in raw or undercooked oysters, can lead to a rapid and extremely fatal infection. Indeed, this one bacterium is responsible for 95 % of all seafood-borne deaths. In addition, the bacterium is capable of entering a preexisting lesion or cut obtained during coastal recreational activities, resulting in potentially fatal wound infections. This brief review, which comprised a presentation made at the Gordon Research Conference on "Oceans and Human Health," reflects over 35 years of research on this bacterium in the author's laboratory. It describes some of the known virulence factors and why males account for ca 85 % of all V. vulnificus cases. It notes the two genotypes now known to exist and how this pathogen enters a dormant, "viable but nonculturable" state during the winter months. Finally, the review discusses how global warming may be causing worldwide increases in the frequency and geographical extent of Vibrio infections.

Highly toxic Microcystis aeruginosa strain, isolated from São Paulo-Brazil, produce hepatotoxins and paralytic shellfish poison neurotoxins.

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777--isolated from Billings's reservoir, São Paulo, Brazil--was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers.The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [L: -ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum.

BACKGROUND: The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. RESULTS: We constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains. CONCLUSIONS: Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

Assessment of shellfish practices in licensed retail food establishments in response to increased Vibrio illnesses in a landlocked area.

Cases of non-cholera Vibrio illness are typically associated with exposure to shellfish from marine coastal areas (U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 2009), not landlocked states such as Colorado. In 2004, a 2.8-fold increase in the incidence of non-cholera Vibrio cases in the Tri-County Health Department (TCHD) jurisdiction of Colorado prompted scrutiny of shellfish practices in local retail food establishments. Forty-three percent of establishments serving raw shellfish in the TCHD jurisdiction were in violation of one or more sections of the Colorado Retail Food Establishment Rules and Regulations (Colorado Department of Public Health and Environment, 2007a). The frequency of violations and the underutilization of safer, post-harvest processed shellfish may result in significant hazards to consumers if these practices continue.

[Preparation and application of the monoclonal antibody against okadaic acid].

An competitive indirect enzyme-linked immunosorbent assay was developed for measurement of okadaic acid (OA), a marine biotoxin associated with red tide. OA was coupled to BSA and OVA by carbodiimide reaction. OA-BSA as immunogen were injected in BALB/c mice. Titres of the antisera against OA were determinated using OA-OVA as coating ligand by ELISA method. The spleen cells of immunized mice were fused with Sp2/O cells. After cloning, four hybridoma cell strains stably produced anti-OA monoclonal antibody were obtained. The monoclonal antibody were produced by the mouse ascites method. The competitive indirect ELISA for okadaic acid in shellfish was established. Under optimal condition, the detection limit of OA was 31.2 ng/ml and the recovery was 87%-112%, with a mean coefficient of variation of 8.1.