Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.
BACKGROUND: From January 2002 to May 2004, 28 puffer puffer, common name for some tropical marine fish of the family Tetraodontidae. The puffers and their allies, the boxfish, the porcupinefish, and the ocean sunfish or headfish, form an odd group (order Tetraodontiformes). fish poisoning (PFP PFP - Plastic Flat Package ) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon The Indian River Lagoon is a series of lagoons and inlets making up a portion of the Atlantic Intracoastal Waterway in the U.S. state of Florida. Its full length extends from Ponce de León Inlet in Volusia County, Florida to Jupiter Inlet in Palm Beach County, Florida (IRL 1. (jargon, chat) IRL - In real life. Generally synonymous with f2f.
2. (language, robotics) IRL - Industrial Robot Language. ) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002.
METHODS: We used the standard mouse bioassay Bioassay
A method for the quantitation of the effects on a biological system by its exposure to a substance, as well as the quantitation of the concentration of a substance by some observable effect on a biological system. (MBA MBA
Master of Business Administration
Noun 1. MBA - a master's degree in business
Master in Business, Master in Business Administration ), receptor binding assay (RBA RBA Rare Bird Alert
RBA Reserve Bank of Australia
RBA Run Book Automation
RBA Rochester Business Alliance
RBA Rights-Based Approach
RBA Royal Brunei Airlines (ICAO code)
RBA Relative Byte Address
RBA relative binding affinity ), mouse neuroblastoma Neuroblastoma Definition
Neuroblastoma is a type of cancer that usually originates either in the tissues of the adrenal gland or in the ganglia of the abdomen or in the ganglia of the nervous system. cytotoxicity assay (MNCA), Ridascreen ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. , MIST Alert assay, HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed , and liquid chromatography-mass spectrometry Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (aka HPLC) with the mass analysis capabilities of mass spectrometry. (LC-MS) to determine the presence of STX STX - Start Of Text , decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL.
RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera viscera /vis·ce·ra/ (vis´er-ah) plural of viscus.
1. The soft internal organs of the body, especially those contained within the abdominal and thoracic cavities. , with concentrations up to 22,104 [micro]g STX equivalents (eq)/100 g tissue (action level, 80 [micro]g STX eq/100 g tissue) in ovaries Ovaries
The female sex organs that make eggs and female hormones.
Mentioned in: Choriocarcinoma
ovaries (ō´v . Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate dinoflagellate
Any of numerous one-celled, aquatic organisms that have two dissimilar flagella and characteristics of both plants (algae) and animals (protozoans). Most are microscopic and marine. P. bahamense as the putative toxin source.
CONCLUSIONS: Associated with fatal paralytic shellfish poisoning Paralytic shellfish poisoning (PSP) is one of the four recognised syndromes of shellfish poisoning (the others being neurologic shellfish poisoning, diarrheal shellfish poisoning and amnesic shellfish poisoning). (PSP (PlayStation Portable) See PlayStation. ) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin saxitoxin /saxi·tox·in/ (sak´si-tok?sin) a powerful neurotoxin synthesized and secreted by certain dinoflagellates, which accumulates in the tissues of shellfish feeding on the dinoflagellates and may cause a severe toxic reaction in puffer fish poisoning (SPFP SPFP Shortest Path First Protocol ) to distinguish it from PFP, which is traditionally associated with tetrodotoxin tetrodotoxin /tet·ro·do·tox·in/ (tet´ro-do-tok?sin) a highly lethal neurotoxin present in numerous species of puffer fish and in certain newts (in which it is called tarichatoxin , and from PSP caused by STXs in shellfish.
KEY WORDS: dinoflagellate, Florida, harmful algae algae (ăl`jē) [plural of Lat. alga=seaweed], a large and diverse group of primarily aquatic plantlike organisms. These organisms were previously classified as a primitive subkingdom of the plant kingdom, the thallophytes (plants that , puffer fish, Pyrodinium bahamense, saxitoxin puffer fish poisoning, saxitoxins, Sphoeroides spp. Environ Health Perspect 114:1502-1507 (2006). doi:10.1289/ehp.8998 available via http://dx.doi.org/ [Online 6 July 2006]
Puffer fish poisoning (PFP) is usually caused by ingestion ingestion /in·ges·tion/ (-chun) the taking of food, drugs, etc., into the body by mouth.
1. The act of taking food and drink into the body by the mouth.
2. of tetrodotoxins (TTXs) found naturally in certain species of puffer fish (Halstead 1967; Mosher A mosher is a person who is crossed between goth/punk/skater they have long hair and listen to music like slipknot and metal music. Some people call them headbangers. At certain music shows they have something called a mosh pit, basically its a fight pit with loads of people bashing each other. and Fuhrmann 1984). In Japan, 20-100 people die annually from PFP, in spite of stringent controls by authorities (Ogura 1971). TTXs can cause fatal human poisoning, which is similar to paralytic shellfish poisoning (PSP) caused by saxitoxins (STXs). PSP is caused by the consumption of toxic shellfish (Shumway 1990) and rarely by fish that have have become toxic after feeding on STX-producing microalgae (Maclean 1979). As well as TTXs, STXs have also been found in at least 12 marine and freshwater puffer fish species in Asia (Ahmed et al. 2001; Kodama et al. 1983; Kungsuwan et al. 1997; Nakamura et al. 1984; Nakashima et al. 2004; Sato et al. 1997, 2000; Zaman et al. 1997), but their bioorigin has not been identified.
TTXs are chemically distinct from STXs, but both neurotoxins produce similar symptoms in mammals because they act on site 1 of the voltage-dependent sodium channel, blocking the influx of sodium into excitable excitable /ex·ci·ta·ble/ (ek-sit´ah-b'l) irritable (1).
1. Capable of reacting to a stimulus. Used of a tissue, cell, or cell membrane.
2. cells and restricting signal transmission along nerve and muscle membranes (Ahmed 1991). The symptoms of traditional PFP from TTXs and of PSP from STXs include tingling and numbness of the mouth, lips, tongue, face, and fingers; paralysis of the extremities; nausea; vomiting; ataxia ataxia (ətăk`sēə), lack of coordination of the voluntary muscles resulting in irregular movements of the body. Ataxia can be brought on by an injury, infection, or degenerative disease of the central nervous system, e.g. ; drowsiness; difficulty in speaking; progressively decreasing ventilatory efficiency; and finally in extreme cases, death by asphyxiation asphyxiation /as·phyx·i·a·tion/ (as-fix?e-a´shun) suffocation; the stoppage of respiration.
Oxygen starvation of tissues. caused by respiratory paralysis (Ahmed 1991; Catterall 1985; Kao 1993).
PFP cases in Europe (Kao 1993) and Mexico (Nunez-Vazquez et al. 2000) have occasionally been reported. In the United States, PFP has been associated with imports of puffer fish [Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. (CDC See Control Data, century date change and Back Orifice.
CDC - Control Data Corporation ) 1996]; rarely have fatalities occurred after the consumption of indigenous puffer fish. In Hawaii, white-spotted puffer fish, Arothron hispidus, were implicated in seven deaths (Ahmed 1991). Until 1974, seven PFP cases in Florida, outside of the Indian River Lagoon (IRL), were caused by the consumption of locally caught "blowfish A secret key cryptography method that uses a variable length key from 32 to 448 bits long. It uses the block cipher method, which breaks the text into 64-bit blocks before encrypting them. " or puffer fish (Ahmed 1991; Benson 1956; Bigler 1999; Hemmert 1974; Mosher and Fuhrmann 1984). These cases included three fatalities, likely from TTX TTX Tetrodotoxin (poison from the Puffer Fish)
TTX Table Top Exercise
TTX True Type Xml
TTX Teletext ; for example, one woman died 45 min after consuming toxic liver from a checkered puffer fish (Sphoeroides testudineus) (Benson 1956). The toxins involved in the previous Florida PFP cases were not characterized, but because PFP is usually associated with TTX, investigators likely assumed that TTX was the cause (Benson 1956; Bigler 1999; Hemmert 1974). Tissues from Florida bandtail (Sphoeroides spengleri), checkered, and southern puffer fish (Sphoeroides nephelus) were found to be lethal in the mouse bioassay (MBA) (Burklew and Morton 1971; Lalone et al. 1963), but, again, the toxins were not determined.
Until January 2002 the harvest and consumption of puffer fish from the IRL was not a risk to public health. Since then (until May 2004), however, 28 PFP cases occurring in Florida (n = 21), New Jersey (n = 3), Virginia (n = 2), and New York (n = 2) caused by puffer fish originating from the IRL were reported (Bodager 2002; CDC 2002a, 2002b). Analyses of toxins from unidentified puffer fish fillet remnants from one of the early 2002 PFP cases in New Jersey revealed STXs (Quilliam et al. 2004), not TTXs, a distinction that alone could not be made on the basis of consumer symptoms or traditional screening methods (i.e., MBA).
During 2002-2004, all PFP cases were linked to puffer fish originating from the northern IRL and the Banana River on Florida's east coast (Figure 1). Except for one case, where puffer fish were commercially harvested and reached a New Jersey fish market, puffer fish were caught recreationally [Bodager 2002; Florida Fish and Wildlife Conservation Commission The Florida Fish and Wildlife Conservation Commission (FWC) is a Florida governmental organization created in 1999 with the purpose of regulating the environment and enforcing environmental legislation in the state of Florida. (FWC FWC Fish and Wildlife Conservation Commission (Florida)
FWC Foster Wheeler Corporation (Clinton, NJ)
FWC Family Winemakers of California
FWC Fresh Water Cooling
FWC Flight Warning Computer ) 2004]. In April 2002, state and federal officials issued health advisories, and the FWC banned puffer fish harvesting in the IRL, a ban that currently remains in effect. In New York on 14 October 2002, two PFP cases were caused from fish caught near Titusville, Florida, but frozen in March 2002 before the harvesting ban (Bodager D, personal communication). This case demonstrated the stability of toxins in puffer fish frozen for almost 9 months.
Because STXs had not previously been identified in Florida's marine waters and their distribution, source, and origin were unknown in April 2002 (Abbott et al. 2003; Landsberg et al. 2002), we initiated an intensive survey of biota biota /bi·o·ta/ (bi-o´tah) all the living organisms of a particular area; the combined flora and fauna of a region.
The flora and fauna of a region. in the IRL. In this article we present a summary from 3 years of monitoring, as well as the first report of the putative toxin source.
Materials and Methods
Field collections. From April 2002 through April 2005, southern, checkered, and bandtail puffer fish (n = 516) were harvested via a range of fishing gear from the original source locations of the PFP incidents in the northern and central IRL (Figure 1). The fish were shipped biweekly or monthly on ice to the FWC's Fish and Wildlife Research Institute (FWRI FWRI Fish and Wildlife Research Institute (St. Petersburg, Florida)
FWRI Florida Wildlife Research Institute ) or to the Food and Drug Administration's (FDA FDA
Food and Drug Administration
n.pr See Food and Drug Administration.
n.pr the abbreviation for the Food and Drug Administration. ) Center for Food Safety and Applied Nutrition The Center for Food Safety and Applied Nutrition (CFSAN, pronounced sif'-san) is the branch of the Food and Drug Administration (FDA) which regulates food, dietary supplements, and cosmetics.
"Food" within the context of FDA is a very broad term with some limitations. Washington Seafood Laboratory and frozen in individual sealable plastic bags until required for toxicity testing.
Live phytoplankton phytoplankton
Flora of freely floating, often minute organisms that drift with water currents. Like land vegetation, phytoplankton uses carbon dioxide, releases oxygen, and converts minerals to a form animals can use. samples were collected routinely with a 62-[micro]m mesh plankton plankton: see marine biology.
Marine and freshwater organisms that, because they are unable to move or are too small or too weak to swim against water currents, exist in a drifting, floating state. net at multiple locations along the IRL; also, a 1-L water bottle was used to directly sample a phytoplankton bloom. Water samples were transported to FWRI at ambient temperature.
Live puffer fish. To determine if puffer fish maintained toxicity once they were removed from the putative toxin source, we kept puffer fish in captivity. We obtained southern puffer fish by rod and line or by seine net from the IRL near Titusville (Atlantic coast) (n = 2) and from Tampa Bay (Gulf coast), Florida (n = 2), and transported them live in ambient seawater to the wet laboratory at FWRI. Southern puffer fish were individually held in covered, 80-L aquaria a·quar·i·a
A plural of aquarium. in 25 psu (practical salinity units) artificial sea water (Instant Ocean; Aquarium Systems, Inc., Mentor, OH) and fed shrimp or squid that originated from nontoxic locations. We measured water quality daily and routinely carried out 30% water exchanges. After several weeks acclimation acclimation /ac·cli·ma·tion/ (ak?li-ma´shun) the process of becoming accustomed to a new environment.
1. , we tested fish skin mucus bimonthly bi·month·ly
1. Happening every two months.
2. Happening twice a month; semimonthly.
1. Once every two months.
2. Twice a month; semimonthly.
n. pl. by lightly anesthetizing the fish [100 ppm tricaine methanesulfonate (MS-222; Sandoz Pharmaceuticals Corp., Basel, Switzerland) in 4 L], placing the fish on a dissection board, and collecting the mucus on a preweighed 47-mm-diameter, glass-fiber filter (Whatman, Clifton, NJ) by gently rubbing the paper along both sides of the body.
Fish care. We conducted research in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals. All fish were treated humanely and with regard for alleviation of suffering, according to the Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996).
Preparation of tissues. Within several weeks of collection, we thawed frozen puffer fish, measured standard lengths and wet weights, and removed skin, liver, stomach, intestinal tract, muscle, and gonads.
Pyrodinium bahamense cultures. We established 11 clonal nonaxenic cultures of P. bahamense from IRL samples, using the micropipette mi·cro·pi·pette
1. A very small pipette used in microinjection.
2. A pipette used to measure very small volumes of liquids.
a pipette for handling small quantities of liquids (up to 1 ml). technique to isolate single cells. We maintained batch cultures in environmental chambers at near-ambient light and temperature conditions (35 microEinsteins/[m.sup.2]/sec, 25[degrees]C) and at salinities of 20-36 psu. Growth media consisted of filtered, autoclaved natural offshore seawater enriched to ES-DK (enriched natural seawater medium modified by D. Kulis) (Kulis D, personal communication; Kokinos and Anderson 1995) levels with the addition of [10.sup.-7] M selenium selenium (səlē`nēəm), nonmetallic chemical element; symbol Se; at. no. 34; at. wt. 78.96; m.p. 217°C;; b.p. about 685°C;; sp. gr. 4.81 at 20°C;; valence −2, +4, or +6. (as sodium selenite).
Toxin detection. At various stages of this survey, we tested puffer fish tissues for STX bioactivity bi·o·ac·tiv·i·ty
The effect of a given agent, such as a vaccine, upon a living organism or on living tissue. using the standard MBA, Ridascreen ELISA (R-Biopharm GmBH, Darmstadt, Germany), MIST Alert (Jellet Biotek, Dartmouth, Canada) PSP kit, and mouse neuroblastoma cytotoxicity assay (MNCA, Neuro-2A) and receptor-binding assay (RBA) [Association of Official Analytical Chemists (AOAC AOAC Association of Official Analytical Chemists (now AOAC International)
AOAC Association of Analytical Communities
AOAC Association of Analytical Chemists
AOAC Always On/Always Connected
AOAC Aero-Optic Evaluation Center ) 1990; Cembella et al. 2003; Jellett et al. 2002; Luckas et al. 2003; Powell and Doucette 1999; Ruberu et al. 2003; Usleber et al. 1991]. We also prepared selected samples for toxin characterization and confirmation by HPLC (Thermo Electron Corporation, San Jose, CA), with postcolumn oxidation and fluorescence detection and liquid chromatography-mass spectrometry (LC-MS) (Waters Corporation, Milford, MA) (Negri et al. 2003; Oshima 1995) using in-house FDA reference standards. We split tissue samples for interlaboratory calibrations and then extracted them by one of two methods. For bioactivity assays, tissue samples were homogenized ho·mog·e·nize
v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es
1. To make homogeneous.
a. To reduce to particles and disperse throughout a fluid.
b. and weighed (wet weight) into glass test tubes. Samples were extracted using 0.1 N HCl, adjusted to pH 2.5-4, boiled for 5 min in a boiling water bath, centrifuged at 3,000 x g for 10 min, and the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. retained for toxin testing. For toxin characterization by HPLC and LC-MS, tissue splits were extracted with 0.1 M aqueous acetic acid, centrifuged, and the supernatants filtered (0.22 [micro]m).
After the initial 2002 saxitoxin puffer fish poisoning (SPFP) events, 11 southern puffer fish were divided into the tissue compartments (listed above), and tissue samples were extracted by boiling in 0.1 N HCl (Washington Seafood Laboratory) and analyzed for toxic activity using three independent methods. MBAs were performed at the Washington Seafood Laboratory; MNCAs were performed at the FDA Gulf Coast Seafood Laboratory; and RBAs were performed at the National Oceanic and Atmospheric Administration Noun 1. National Oceanic and Atmospheric Administration - an agency in the Department of Commerce that maps the oceans and conserves their living resources; predicts changes to the earth's environment; provides weather reports and forecasts floods and hurricanes and National Ocean Service Center for Coastal Environmental Health and Biomolecular Research.
Clonal cultures and natural bloom samples of P. bahamense were filtered onto 25-mm glass-fiber filters (Whatman) or centrifuged at 3,000 x g for 5 min and then extracted. Puffer fish mucus or Pyrodinium samples on filters were homogenized in 0.1 N HCl using a ground-glass tissue grinder and treated as above. Pyrodinium extracts were tested for toxicity by ELISA and MBA and characterized for toxin profile using HPLC and LC-MS. Puffer fish mucus was tested for toxicity by ELISA.
Electron microscopy. We prepared natural field samples or clonal cultures of P. bahamense for the scanning electron microscope scan·ning electron microscope
n. Abbr. SEM
An electron microscope that forms a three-dimensional image on a cathode-ray tube by moving a beam of focused electrons across an object and reading both the electrons scattered by the object and (SEM) using standard fixation methods (Truby 1997). Pyrodinium samples were added to unacidified Lugol's at a dilution of 1:100 in suspension, collected onto a 5-[micro]m polycarbonate A category of plastic materials used to make a myriad of products, including CDs and CD-ROMs. filter, secondarily fixed with 4% paraformaldehyde paraformaldehyde: see formaldehyde. for 20 min, washed with water, dehydrated de·hy·drate
v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates
1. To remove water from; make anhydrous.
2. To preserve by removing water from (vegetables, for example). in an ethanol series followed by a freon series, critical-point-dried using carbon dioxide, mounted onto aluminum stubs using carbon adhesive tape, sputter-coated with gold/palladium, and photographed with a Cambridge Stereoscan 240 SEM (Cambridge Instruments, Cambridge, UK).
Puffer fish toxin analyses. During 36 months of continuous monitoring after the initial SPFP events, STXs were routinely detected in the skin, muscle, viscera, and gonads of 516 puffer fish (southern, n = 402; checkered, n = 105; bandtail, n = 9), which tested toxic in MBA, RBA, Ridascreen ELISA, and MIST Alert assays. By ELISA, maximum STX levels in the muscle fillet were well above the action level [80 [micro]g STX equivalents (eq)/100 g tissue] in southern puffer fish (maximum, 14,571 [micro]g STX eq/100 g tissue, mean = 938.4) and just over the action limit in bandtail (maximum, 364.5 [micro]g STX eq/100 g tissue; mean, 121.7) and checkered puffer fish (maximum, 104.3 [micro]g STX eq/100 g tissue; mean, 6.9) (Table 1). Maximum STX concentrations in the liver of southern and checkered puffer fish were 1,443 and 51.1 [micro]g STX eq/100 g tissue, respectively. The highest tissue concentration, 22,104 [micro]g STX eq/100 g tissue, was measured in the ovaries of a southern puffer fish (data not shown).
All three assays (MBA, MNCA, and RBA) confirmed elevated concentrations of toxic activity in the muscle compared to the liver (5- to 20-fold) of 11 southern puffer fish (Table 2). By MBA, MNCA, and RBA, ranges of STX concentrations in muscle were 197-5,264 (mean [+ or -] SD, 2,302.3 [+ or -] 1,539.3), 120-2,294 (957.7 [+ or -] 659.5), and 198-6,091 (2,439 [+ or -] 1,995.3) [micro]g STX eq/100 g tissue, respectively. By MBA, MNCA, and RBA, STX concentrations in liver were 83-1,034 (mean [+ or -] SD, 282.6 [+ or -] 261.5), 60-420 (169.1 [+ or -] 106.2) and 16-711 (223.1 [+ or -] 186.5) [micro]g STX eq/100 g tissue, respectively (Table 2).
Skin mucus of IRL southern puffer fish held captive for 1 year was highly toxic (2,407-9,039 [micro]g STX eq/100 g) compared with that of Florida Gulf Coast southern puffer fish (6.25-140 [micro]g STX eq/100 g). Over a period of at least 6 months, STX levels in the IRL southern puffer fish fluctuated but remained at highly toxic concentrations.
Toxin profiles in unconsumed puffer fish fillets (n = 4) from a 2004 PFP event were confirmed by HPLC (Figure 2A,B) and LC-MS (Figure 2C-F) to be STX (92.4% [+ or -] 3.1), decarbamoyl saxitoxin (dcSTX; 6.9% [+ or -] 2.4), and N-sulfocarbamoyl B1 toxin (B1; 0.7% [+ or -] 0.7) as originally found in a 2002 PFP case (Quilliam et al. 2004).
We also detected TTX (quantified by MBA and confirmed by LC-MS) in IRL checkered puffer fish (n = 3) at concentrations of 1,553 [+ or -] 919 and 53,700 [+ or -] 19,212 [micro]g TTX/100 g in the muscle and liver, respectively (Figure 3A-D A-D
Advance-Decline, or measurement of the number of issues trading above their previous closing prices less the number trading below their previous closing prices over a particular period. ).
Pyrodinium bahamense toxin analyses. All clonal cultures (n = 11) and natural bloom samples (n = 2) (> 3 million cells/L) of P. bahamense (Figure 4) obtained from the IRL tested positive for STX by HPLC, LC-MS, Ridascreen ELISA, MIST Alert, and RBA assays. Toxin concentrations for P. bahamense isolates (n = 11) ranged from 1.68 to 25.57 pg STX eq/cell (as determined by ELISA). Further analysis of five of these isolates using HPLC determined that the toxin profile was composed of B1 (91.1% [+ or -] 2.2, mean [+ or -] SD) and STX (8.9% [+ or -] 2.2), with integrated toxicity values ranging from 2.02 to 12.74 pg STX eq/cell. The HPLC toxin profile of a 2002 bloom sample at 3.28 pg STX eq/cell was composed of STX (26%), B1 (73%), and dcSTX (1%) (Figure 5A,B).
PFP cases have been associated with STXs in Asia (Ahmed et al. 2001), but the IRL incidents are the first in which STX poisoning has been confirmed in puffer fish originating in the United States (Quilliam et al. 2004). The high and low concentrations of STXs and TTX, respectively, in the muscle of IRL puffer fish are similar to those found in Philippine (Sato et al. 2000) and Japanese (Kodama et al. 1984) puffer fish, although in the latter, visceral toxicity from TTX is high and fish-poisoning incidents usually occur after consumption of fillet(s) contaminated due to improper preparation. Unlike the tissue distribution of TTXs reported previously in various puffer fish species (Kodama et al. 1984), STXs in IRL southern puffer fish have been consistently much higher in the muscle than in the liver and, in many individual fish, were more than two orders of magnitude above the action limit. Therefore, even careful preparation of IRL puffer fish fillets would not prevent intoxication in consumers. Interestingly, the confirmation of extremely high concentrations of TTX in the liver of checkered puffer fish suggests that the earlier-reported fatality from the consumption of this species in south Florida (Benson 1956) was likely caused by this toxin and not STX.
The MBA, the traditional screening method for PFP, does not distinguish between STXs and TTXs. New reports in Asia (Ahmed et al. 2001; Nakashima et al. 2004; Sato et al. 2000) have found both toxin groups co-occurring in puffer fish species previously thought to contain only TTX. Both our results and these reports suggest that STXs in puffer fish may be more widespread than previously thought; therefore, comprehensive analytical assessments of PFP incidents are needed to distinguish TTX from STX. We propose that the food-poisoning syndrome caused by intoxication from STX exposure from fish should be characterized as SPFP to distinguish it from PFP, which is caused by--but not always verified to be from--TTX, and to distinguish SPFP from PSP associated with STXs in shellfish.
In a 1960s toxicity study of IRL southern puffer fish [erroneously identified by Lalone et al. (1963) as northern puffer fish, Sphoeroides maculatus, which are not found in the IRL and occur only as far south as Jacksonville, FL (Shipp and Yerger 1969a, 1969b; Tremain and Adams, 1995)], muscle was demonstrated to be toxic to mice by intraperitoneal injection. However, the toxins in these puffer fish samples were not characterized. Of the tissues investigated in that study, including skin, liver, muscle, and testes testes
Male reproductive organs (see reproductive system). Humans have two oval-shaped testes 1.5–2 in. (4–5 cm) long that produce sperm and androgens (mainly testosterone), contained in a sac (scrotum) behind the penis. or ovary ovary, ductless gland of the female in which the ova (female reproductive cells) are produced. In vertebrate animals the ovary also secretes the sex hormones estrogen and progesterone, which control the development of the sexual organs and the secondary sexual , the muscle was the most lethal to mice, similar to the pattern seen today. Although this anecdotal evidence suggests that southern puffer fish may have been mildly toxic from STX in the IRL for the past 45 years, there has been no indication that toxin levels were even close to the order of magnitude A change in quantity or volume as measured by the decimal point. For example, from tens to hundreds is one order of magnitude. Tens to thousands is two orders of magnitude; tens to millions is three orders of magnitude, etc. observed since 2002 nor was the FDOH FDOH Florida Department of Health informed of any poisoning incidents from this area prior to this time.
Globally, human food-poisoning incidents from STX exposure are usually caused by toxic marine shellfish (Kao 1993) that filter-feed on STX-producing microalgae. PSP can be fatal (Kao 1993), but the successful implementation of programs monitoring STX-producing microalgae and STXs in shellfish has helped minimize the risk of toxin exposure to humans. In marine waters, PSP is caused by toxic dinoflagellates dinoflagellates
minute aquatic protozoa; they produce red pigment and toxins which are taken up by shellfish without apparent ill effect, but the toxin is not metabolized and the shellfish may poison animals if eaten. , where STXs are produced by more temperate Alexandrium species and Gymnodinium catenatum and by tropical Pyrodinium bahamense var. compressum (Kao 1993). PSP in the United States has been limited to New England and the Pacific West Coast, including Alaska, and has only been associated with STXs produced by temperate Alexandrium spp. in these areas (Gessner 2000).
The epidemiology of PSP incidents is related to the global distribution of the various STX-producing species and their toxigenic toxigenic /tox·i·gen·ic/ (tok?si-jen´ik)
1. producing or elaborating toxins.
2. derived from or containing toxins.
Producing a poison; toxicogenic. strains. PSP outbreaks due to P. bahamense have caused more fatalities than any other microalgal species known (Usup and Azanza 1998). In 1987, PSP associated with P. bahamense var. compressum in Champerico, Guatemala, hospitalized at least 187 individuals and resulted in 26 fatalities (Rodrigue et al. 1990). Before 1996, 1,768 cases of PSP with 107 deaths had been reported in the Philippines, mostly attributable to P. bahamense var. compressum (Babaran et al. 1998). These fatalities were largely due to the sudden appearance of P. bahamense in areas previously unknown to contain toxic species, because monitoring activities were not in place or because hospital facilities had not treated people in these previously unaffected areas (Kao 1993).
In the present study we confirm unequivocally that puffer fish are a primary reservoir of STXs in marine waters in Florida, and we implicate for the first time the tropical western Atlantic dinoflagellate P. bahamense as the source of toxicity. We found the STX profile of P. bahamense isolates from Florida to be similar to, but proportionately different from, the toxin profile of southern puffer fish fillet (Etheridge et al. 2006; Quilliam et al. 2004), and we identified P. bahamense as the putative source of the STXs. Confirmatory toxin-transfer studies from Pyrodinium via shellfish to puffer fish are in progress. Although many temperate marine Alexandrium species, Gymnodinium catenatum, and a few freshwater cyanobacteria cyanobacteria (sī'ənōbăktĭr`ēə, sī-ăn'ō–) or blue-green algae, photosynthetic bacteria that contain chlorophyll. species produce STXs (Kodama 2000), these organisms have not been found in the IRL.
In addition to the Caribbean and Gulf coasts of Mexico, bioluminescent bi·o·lu·mi·nes·cence
Emission of visible light by living organisms such as the firefly and various fish, fungi, and bacteria.
bi P. bahamense blooms are found only along Florida's Atlantic and Gulf coasts (Badylak et al. 2004; Phlips et al. 2004; Steidinger et al. 1980). However, until the IRL SPFP incidents, the Atlantic/Caribbean P. bahamense var. bahamense was not known to be toxic (Steidinger et al. 1980), unlike the Pacific P. bahamense var. compressum found in Asia and the Pacific Coast of Central America (Rodrigue et al. 1990; Usup and Azanza 1998; Vargas-Montero and Freer 2004). The Atlantic P. bahamense var. bahamense was separated from the Pacific P. bahamense var. compressum based on morphologic criteria and evident lack of toxicity in the former variety (Steidinger et al. 1980). Based on our initial findings, we are testing the hypothesis that this varietal distinction may no longer be valid and that P. bahamense is all one species.
Florida has many toxigenic marine algal algal
pertaining to or caused by algae.
is very rare but systemic and udder infections are recorded. See protothecosis.
the algae Prototheca trispora and P. species, but none were known to produce STXs (Steidinger et al. 1999). It is conceivable that STXs might have appeared in the IRL because of one of several scenarios: a) toxigenic populations of Pyrodinium have been introduced; b) ecologic conditions have changed and have induced toxicity in a variety that was previously nontoxic; c) toxic Pyrodinium was present but produced toxins at undetectable concentrations; or d) ecologic conditions have changed and increased the food-web exposure of susceptible biota to toxins. We believe that c is the most likely scenario. In the IRL there is a history of Pyrodinium (Badylak et al. 2004; Phlips et al. 2004), and as mentioned previously, there is a historical precedent for low-level toxicity in puffer fish.
In the past few years, the northern IRL has experienced a number of unusual events: dolphin, manatee, fish, and horseshoe crab mortalities; increased tumor incidence in hard clams; diseased shrimp; and reductions in the natural recruitment of and increases in the hatchery hatchery
a commercial establishment dedicated to the hatching of bird eggs to provide day old chicks and poults to the poultry industry.
the contents of unfertilized eggs. Used in petfood manufacture. losses of hard clams (Bossart et al. 2003; Landsberg et al. 2002; Landsberg and Kiryu 2005). To what extent, if at all, these events are linked to the emerging issue of toxic P. bahamense blooms remains undetermined. The significant risk of SPFP and PSP from saxitoxins in the IRL has been assessed and management plans implemented accordingly. Thus far, routine monitoring by Florida state agencies has determined that STX levels in shellfish, principally hard clams (Mercenaria spp.), are not a significant risk to public health (Landsberg et al. 2005). The extreme toxicity of puffer fish fillet, well above the action level, emphasizes the danger that puffer fish pose to the public and supports the permanent ban on their harvest in this area (FWC 2004).
The widespread implications for public health incidents from the tropical western Atlantic P. bahamense remain unknown. Public health officials and natural resource managers should be aware of these new findings and remain vigilant to examine any potential association between the co-occurrence of this species throughout its range and the appearance of toxic food-poisoning incidents.
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a series of unicellular algae, microscopic in size, with cell walls containing silica. Members of the family Diatomaceae. Their remains accumulate as geological deposits and are mined. See diatomaceous earth. in a subtropical sub·trop·i·cal
Of, relating to, or being the geographic areas adjacent to the Tropics.
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a. A multitude; a throng.
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2. People who are followers, not leaders.
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Jan H. Landsberg, (1) Sherwood Hall, (2) Jan N. Johannessen, (3) Kevin D. White, (4) Stephen M. Conrad, (2) Jay P. Abbott, (1) Leanne J. Flewelling, (1) R. William Richardson, (1) Robert W. Dickey, (5) Edward L.E. Jester, (5) Stacey M. Etheridge, (2) Jonathan R. Deeds, (2) Frances M. Van Dolah, (6) Tod A. Leighfield, (6) Yinglin Zou, (7) Clarke G. Beaudry, (4) Ronald A. Benner, (2) Patricia L. Rogers, (2) Paula S. Scott, (1) Kenji Kawabata, (1) Jennifer L. Wolny, (1,8) and Karen A. Steidinger (1,8)
(1) Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, Florida St. Petersburg (often shortened to St. Pete) is a city in Pinellas County, Florida, United States. The city is known as a vacation destination for North American and European vacationers, as well as a politically important battleground in U.S. Presidential politics. , USA; (2) Food and Drug Administration, Center for Food Safety and Applied Nutrition, Laurel, Maryland, USA; (3) Food and Drug Administration, Office of the Commissioner, Rockville, Maryland, USA; (4) Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, Maryland College Park is a city in Prince George's County, Maryland, USA. The population was 24,657 at the 2000 census. It is best known as the home of the University of Maryland, College Park, and since 1994 the city has also been home to the "Archives II" facility of the U.S. , USA; (5) Food and Drug Administration, Center for Food Safety and Applied Nutrition, Gulf Coast Seafood Laboratory, Dauphin Island, Alabama Dauphin Island, Alabama is a town in Mobile County, Alabama, on a barrier island also named Dauphin Island. As of the 2000 census, the population of the town is 1,371. It is included in the Mobile metropolitan statistical area. , USA; (6) National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina, USA; (7) Key Laboratory of Science and Engineering for Marine Ecology and Environment, First Institute of Oceanography oceanography, study of the seas and oceans. The major divisions of oceanography include the geological study of the ocean floor (see plate tectonics) and features; physical oceanography, which is concerned with the physical attributes of the ocean water, such as , State Oceanic Administration, Qingdao, China; (8) Florida Institute of Oceanography, University of South Florida
• • [ , St. Petersburg, Florida, USA
Address correspondence to J.H. Landsberg, Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 Eighth Ave. SE, St. Petersburg, FL 33701 USA. Telephone: (727) 896-8626. Fax: (727) 893-9840. E-mail: email@example.com
We thank L. Sebastian, R. Paperno, D. Adams, D. Tremain, S. Fisk, S. Stahl, S. Cook, J. D'Urso, and A. Shurtleff, Florida Fish and Wildlife Conservation Commission (FWC), for technical assistance and D. Bodager and G. Jackow, Florida Department of Health (FDOH), for specimen collection.
Funding or support for this research was provided by the FWC, FDOH, U.S. Food and Drug Administration, Centers for Disease Control and Prevention, and the National Oceanic and Atmospheric Administration (NOAA NOAA
National Oceanic and Atmospheric Administration
Noun 1. NOAA - an agency in the Department of Commerce that maps the oceans and conserves their living resources; predicts changes to the earth's environment; ). This article is a result of research partially funded by the NOAA Coastal Ocean ECOHAB ECOHAB Ecology and Oceanography of Harmful Algal Blooms Program under award #NA03NOS4780196 to the FWC (ECOHAB contribution #152).
The authors declare they have no competing financial interests.
Received 11 January 2006; accepted 5 July 2006.
Table 1. Comparison of saxitoxin concentrations ([micro]g STX eq/100 g tissue) in muscle and liver of IRL puffer fish species by ELISA. Muscle Puffer fish species No. Mean [+ or -] SD Maximum Southern 402 938.4 [+ or -] 1,418 14,571 Checkered 105 6.9 [+ or -] 11.4 104.3 Bandtail 9 121.7 [+ or -] 117.9 364.5 Liver Puffer fish species No. Mean [+ or -] SD Maximum Southern 55 265.6 [+ or -] 393 1,443 Checkered 3 20.3 [+ or -] 27.1 51.1 Bandtail 0 -- -- Table 2. Comparison of saxitoxin-like activity levels ([micro]g STX dihydrochloride eq/100 g tissue) by LC-MS in muscle and liver of southern puffer fish (S. nephelus) collected from the IRL after the first SPFP cases in 2002. MBA RBA MNCA Fold Fold Fold Fish Muscle Liver diff Muscle Liver diff Muscle Liver diff 1 5,264 1,034 5.1 4,136 711 5.8 2,294 420 5.5 2 4,697 376 12.5 6,091 304 20.0 1,230 280 4.4 3 2,986 242 12.3 2,433 280 8.7 1,947 160 12.2 4 2,804 203 13.8 1,423 147 9.7 1,100 120 9.2 5 2,564 149 17.2 5,253 297 17.7 844 110 7.7 6 2,153 135 15.9 2,911 173 16.8 790 240 3.3 7 1,970 263 7.5 2,257 142 15.9 750 150 5.0 8 1,216 254 4.8 805 154 5.2 350 140 2.5 9 1,098 221 5.0 1,089 180 6.1 480 110 4.4 10 376 83 4.5 198 16 12.4 630 70 9.0 11 197 149 1.3 231 50 4.6 120 60 2.0 Fold diff indicates fold difference of muscle compared with liver.