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Kim S.,Smithsonian Environmental Research Center | Kim S.,Kongju National University | Bachvaroff T.R.,Smithsonian Environmental Research Center | Handy S.M.,Us Fda Center For Food Safety And Applied Nutrition | Delwiche C.F.,University of Maryland College Park
Molecular Biology and Evolution | Year: 2011

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5′ UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species. © 2010 The Author.

Petitpas C.M.,University of Massachusetts Dartmouth | Turner J.T.,University of Massachusetts Dartmouth | Deeds J.R.,Us Fda Center For Food Safety And Applied Nutrition | Keafer B.A.,Woods Hole Oceanographic Institution | And 5 more authors.
Deep-Sea Research Part II: Topical Studies in Oceanography | Year: 2014

As part of the Gulf of Maine Toxicity (GOMTOX. 11Gulf of Maine TOXicity (GOMTOX) project, we determined Alexandrium fundyense abundance, paralytic shellfish poisoning (PSP) toxin levels in various plankton size fractions, and the community composition of potential grazers of A. fundyense in plankton size fractions during blooms of this toxic dinoflagellate in the coastal Gulf of Maine and on Georges Bank in spring and summer of 2007, 2008, and 2010. PSP toxins and A. fundyense cells were found throughout the sampled water column (down to 50. m) in the 20-64. μm size fractions. While PSP toxins were widespread throughout all size classes of the zooplankton grazing community, the majority of the toxin was measured in the 20-64. μm size fraction. A. fundyense cellular toxin content estimated from field samples was significantly higher in the coastal Gulf of Maine than on Georges Bank. Most samples containing PSP toxins in the present study had diverse assemblages of grazers. However, some samples clearly suggested PSP toxin accumulation in several different grazer taxa including tintinnids, heterotrophic dinoflagellates of the genus Protoperidinium, barnacle nauplii, the harpacticoid copepod Microsetella norvegica, the calanoid copepods Calanus finmarchicus and Pseudocalanus spp., the marine cladoceran Evadne nordmanni, and hydroids of the genus Clytia. Thus, a diverse assemblage of zooplankton grazers accumulated PSP toxins through food-web interactions. This raises the question of whether PSP toxins pose a potential human health risk not only from nearshore bivalve shellfish, but also potentially from fish and other upper-level consumers in zooplankton-based pelagic food webs. © 2013 Elsevier Ltd.

Tallent S.M.,Us Fda Center For Food Safety And Applied Nutrition | DeGrasse J.A.,Us Fda Center For Food Safety And Applied Nutrition | Wang N.,University of Illinois at Urbana - Champaign | Mattis D.M.,University of Illinois at Urbana - Champaign | Kranz D.M.,University of Illinois at Urbana - Champaign
Applied and Environmental Microbiology | Year: 2013

Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vβ domain of the T-cell receptor (Vβ-TCR) or polyclonal antibodies. The binding affinity of the Vβ-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vβ-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vβ-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample. © 2013, American Society for Microbiology.

Diaz-Amigo C.,Us Fda Center For Food Safety And Applied Nutrition
Food Analytical Methods | Year: 2010

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose. © 2009 Springer Science+Business Media, LLC.

Diaz-Amigo C.,Us Fda Center For Food Safety And Applied Nutrition
Food Analytical Methods | Year: 2010

Milk and milk derivatives are common ingredients in food products. Undeclared milk is one of the leading causes of recalls in many countries, including the USA, and cases of allergic reactions have been reported due to unexpected exposures. There are commercial enzyme-linked immunosorbent assay (ELISA) kits available to the food industry to comply with the law by ensuring label accuracy and to identify potential sources of cross-contact. These kits are also used by regulatory agencies as part of their compliance programs. However, none of the commercial ELISAs for milk have been validated. Performance of ELISA kits for food allergens is affected by matrix, food processing, and stability and solubility of target proteins, among others factors. The performance of different commercial kits for milk allergens was evaluated by comparing a standard [National Institute of Standards and Technology (NIST) SRM #1549] spiked in wheat flour. We also compared the effect of food processing on detectability of milk proteins from incurred peanut butter cookies baked at various times. Kits differed in their ability to detect heat-treated milk proteins in baked cookies. Immunoblots clearly showed differences in antibody specificities and in their ability to detect proteins in processed foods. Factors such as undefined antibody specificity and differences in sample extraction solutions, materials used for calibrators, and reporting units contribute to variability of results among test kits and, hence, to increased uncertainty regarding the most appropriate use of the kits. Moreover, the use of incurred vs. spiked samples may affect protein recovery and, therefore, jeopardize the quantitative nature of the kit. © 2009 Springer Science+Business Media, LLC.

Braid H.E.,University of Guelph | Braid H.E.,Auckland University of Technology | Deeds J.,Us Fda Center For Food Safety And Applied Nutrition | DeGrasse S.L.,Us Fda Center For Food Safety And Applied Nutrition | And 3 more authors.
Marine Biology | Year: 2012

In fall of 2009, several mass strandings of Humboldt squid (Dosidicus gigas) occurred on Vancouver Island (49°7′60N 125°54′0W). Morphological dissections coupled with DNA barcoding of stomach contents revealed Sardinops sagax (Pacific sardine) and Clupea pallasii (Pacific herring) as their primary prey. Plastic nurdles, fishing line, bull kelp, eelgrass, and a guillemot feather were also discovered. The primary prey, Pacific sardines and Pacific herring, are known to bioaccumulate paralytic shellfish toxins (PSTs); additionally, both PSTs and domoic acid (DA) have been implicated in other mass strandings. Therefore, stomach contents, and other tissues when possible, were tested for PSTs and DA. Testing revealed DA concentrations below regulatory guidance levels for human consumption, yet PSTs were well in excess. Though we cannot conclude that PSTs were the definitive cause of the strandings, our findings are the first report of PSTs in D. gigas. © 2011 Springer-Verlag.

Anderson M.,Rti International | Jaykus L.-A.,North Carolina State University | Beaulieu S.,Rti International | Dennis S.,Us Fda Center For Food Safety And Applied Nutrition
Food Control | Year: 2011

Foodborne disease outbreaks and cases associated with fresh produce have increased over the past decade. In developing approaches to prevent or reduce illnesses from consumption of contaminated produce, new tools are needed to identify and prioritize appropriate efforts. The purpose of this study was to develop a semi-quantitative risk ranking tool (Pathogen-Produce Pair Attribution Risk Ranking Tool, or P3ARRT) to rank the relative public health impact of pathogen-produce commodity combinations, based on explicit data-driven criteria. To identify candidate pathogen-commodity pairs, a database was created that included all published reports of fresh produce-associated outbreaks in the United States. A total risk score was calculated for each pathogen-commodity pair as the summation of nine criteria scores multiplied by the respective criteria weighting. A total of 53 pathogen-produce commodity pairs were included in the risk ranking, and based on scenario and sensitivity analyses, enterohemorrhagic E. coli in leafy greens consistently ranked first, followed by Salmonella spp. in tomatoes, and Salmonella spp. in leafy greens. The P3ARRT model provides a systematic, transparent, and customizable tool with which to prioritize produce pathogen-commodity pairs for further, more rigorous risk assessment modeling and evaluation efforts. The tool is available to the public at © 2011 Elsevier Ltd.

Dickey R.W.,Us Fda Center For Food Safety And Applied Nutrition | Plakas S.M.,Us Fda Center For Food Safety And Applied Nutrition
Toxicon | Year: 2010

Ciguatera fish poisoning is a seafood-borne illness caused by consumption of fish that have accumulated lipid-soluble ciguatoxins. In the United States, ciguatera is responsible for the highest reported incidence of food-borne illness outbreaks attributed to finfish, and it is reported to hold this distinction globally. Ciguatoxins traverse the marine food web from primary producers, Gambierdiscus spp., to commonly consumed fish in tropical and subtropical regions of the world. Ciguatoxins comprise 12 known congeners among Caribbean and tropical Atlantic fish and 29 reported congeners among Pacific fish. Expanding trade in fisheries from ciguatera-endemic regions contributes to wider distribution and increasing frequency of disease among seafood consumers in non-endemic regions. Ciguatoxins produce a complex array of gastrointestinal, neurological and cardiological symptoms. Treatment options are very limited and supportive in nature. Information derived from the study of ciguatera outbreaks has improved clinical recognition, confirmation, and timely treatment. Such studies are equally important for the differentiation of ciguatoxin profiles in fish from one region to the next, the determination of toxicity thresholds in humans, and the formulation of safety limits. Analytical information from case and outbreak investigations was used to derive Pacific and Caribbean ciguatoxin threshold contamination rates for adverse effects in seafood consumers. To these threshold estimates 10-fold safety factors were applied to address individual human risk factors; uncertainty in the amount of fish consumed; and analytical accuracy. The studies may serve as the basis for industry and consumer advisory levels of 0.10. ppb C-CTX-1 equivalent toxicity in fish from the tropical Atlantic, Gulf of Mexico, Caribbean, and 0.01. ppb P-CTX-1 equivalent toxicity in fish from Pacific regions. © 2009.

Newsome G.A.,Nova Research Inc. | Ackerman L.K.,Us Fda Center For Food Safety And Applied Nutrition | Johnson K.J.,U.S. Navy
Journal of the American Society for Mass Spectrometry | Year: 2016

Post-plasma ambient desorption/ionization (ADI) sources are fundamentally dependent on surrounding water vapor to produce protonated analyte ions. There are two reports of humidity effects on ADI spectra. However, it is unclear whether humidity will affect all ADI sources and analytes, and by what mechanism humidity affects spectra. Flowing atmospheric pressure afterglow (FAPA) ionization and direct analysis in real time (DART) mass spectra of various surface-deposited and gas-phase analytes were acquired at ambient temperature and pressure across a range of observed humidity values. A controlled humidity enclosure around the ion source and mass spectrometer inlet was used to create programmed humidity and temperatures. The relative abundance and fragmentation of molecular adduct ions for several compounds consistently varied with changing ambient humidity and also were controlled with the humidity enclosure. For several compounds, increasing humidity decreased protonated molecule and other molecular adduct ion fragmentation in both FAPA and DART spectra. For others, humidity increased fragment ion ratios. The effects of humidity on molecular adduct ion fragmentation were caused by changes in the relative abundances of different reagent protonated water clusters and, thus, a change in the average difference in proton affinity between an analyte and the population of water clusters. Control of humidity in ambient post-plasma ion sources is needed to create spectral stability and reproducibility. [Figure not available: see fulltext.] © 2015 American Society for Mass Spectrometry.

Newsome G.A.,Nova Research Inc. | Ackerman L.K.,Us Fda Center For Food Safety And Applied Nutrition | Johnson K.J.,U.S. Navy
Analytical Chemistry | Year: 2014

Unstable explosive hexamethylene triperoxide diamine (HMTD) is dangerous in quantity and benefits from the minimal sampling handling associated with atmospheric pressure chemical ionization for mass spectral analysis. Seasonal variation observed in HMTD mass spectra suggested a humidity dependence. Therefore, direct analysis in real time (DART) ionization mass spectra were acquired at a range of humidity values. An enclosure was designed to fit around the ion source and mass spectrometer inlet at atmospheric pressure. The enclosure was supplied with controlled amounts of humidified air from a test atmosphere generator to create programmable conditions for ambient analysis. The relative abundance and fragmentation of analyte ions were observed to change reliably with changing humidity values and, to a lesser degree, temperature. Humidity at such plasma-based ion sources should be regulated to avoid ∼90% shifts in relative ion abundance and provide stability and reproducibility of HMTD analysis. (Figure Presented). © 2014 American Chemical Society.

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