Toxin and toxic organisms causing food poisoningiocwestpac.org/files/upload_manual/Lecture 1.pdf · 2014-11-12 · Toxin and toxic organisms causing food poisoning Dao Viet Ha Head

Toxin and toxic organisms

causing food poisoning

Dao Viet HaHead of Biochemistry Department Institute of Oceanography01 Cau Da – Nha Trang - Vietnam

WESTPAC workshop 2010.Dec.13

Marine products especially fish & shellfish: Important food resources in the Western Pacific &

the ASEAN countries

Aquacultural activities in Cam Ranh Bay, Khanh Hoa (Photo by Fukuyo, 2005)

Aquaculture becomes more important

Two types of toxic organism in aquatic environment

1.Toxic by taking toxic organisms such as microalgae by feeding activity (well-known)

example: FishBivalve…

2. Toxic by its own toxin production (unknown) (either symbiotic bacteria or encode in gene)

example: PufferBlue-ringed octopusGoby fishJelly fishConusCrab…


1. Toxic by taking toxic organisms such as microalgae by feeding activity


2. Toxic by its own toxin production (either symbiotic bacteria or encode in gene)

example: PufferBlue-ringed octopusGoby fishJelly fishConusCrab…

Toxins in HAB organisms:

human poisoning

Characters of the accumulation type1.Toxic by taking toxic organisms such as microalgae by

feeding activity1) Toxin accumulation occurs during blooming of causative

microalgae 2) Toxins are excreted constantly, even during accumulation.3) Speed of accumulation and excretion is different

depending on species. 4) All filter feeding organisms have potential to become toxic

(cases of poisoning by a sea squirt were reported)5) Depending on microalgae fed, toxins in organisms differ;

Naturally poisoning caused by the organisms vary.PSP (paralytic shellfish poisoning)DSP (diarrehtic shellfish poisoning)ASP (amnesic shellfish poisoning)CFP (ciguatera fish poisoning) etc.







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Toxin

Origin of toxins are unicellular microalgae

Paralytic shellfish poison (PSP) Diarrhetic shellfish poison (DSP) Amnesic shellfish poison (ASP)Neurotoxic shellfish poison (NSP) Azaspiracid (AZA)

Toxin accumulation

PSP Toxin accumulates in relation to the abundance of causative plankton.Toxins are released, even during toxic plankton presence.

Max PSP toxin

North America

South China

Each shellfish species has different toxin accumulation character.

Scallop

Mussel Toxin excretion pattern of scallop varies from that of mussel:

Scallop keeps toxins much longer than mussel.

Certain amount of toxins remain in scallop very long time.

Toxin excretion

Toxin excretion

Toxin excretion: oyster, mussel: fast; clam, scallop: slow







toxi

city

(MU

/g)

May June July

Scallop

Oyster

Mussel

tunicate

Toxin accumulation speed varies species by species.

Filter feeding organisms, such as scallop, mussel and sea skirt become toxic during toxic plankton blooming.

PSP plankton bloom





(cases of poisoning by a tunicate were reported)5) Depending on microalgae fed, toxins in organisms differ;


Toxins caused human poisoning:1. Fish vector: Ciguatoxin,

2. Shellfish vector:

Paralytic shellfish poison (PSP) (saxitoxin and its derivatives) Diarrhetic shellfish poison (DSP) (dinophysistoxin, pectenotoxin, yessotoxin) Amnesic shellfish poison (ASP) (domoic acid) Neurotoxic shellfish poison (NSP) (brevetoxin) Azaspiracid shellfish poison (AZP)




Gambierdiscus toxicus

Human poisoning: Ciguatera Fish Poisoning

is most frequently reported seafood-toxin illness in the world.

illness results from eating reef fish containing ciguatoxins derived from benthic dinoflagellates of the genus Gambierdiscus growing in association with macroalgae in coral reefs in tropical and subtropical climates.

toxins are transferred through the food web by herbivorous and carnivorous fish.

O

O

O

OO

O O

OO

O

OOO

HO

H

OH

HH

OH

HH

HH

H HH H

HOH3C H

H3C H HCH3OH

H

OHCH3H3C

HHHHH

H

5

1

15

30

40

50

Characters of toxins responsible for ciguatera

Ciguatoxin main toxin for ciguatera fat soluble

Maitotoxin water soluble toxin

CTX activates the voltage-gated sodium channels in cell membranes which increases sodium ion permeability and depolarizes the nerve cell causing an array of neurological symptoms

under-reporting may be considerable; between10 and 50 thousand people per year are estimated to suffer from CFP

in some patients chronic symptoms may last for months

Human poisoning: Ciguatera Fish Poisoning

Ciguatera: 0.05 µg (mild symptom)

Acute: gastrointestinal, neurological and cardiovascular signs.

Symptoms (within a few hours of contaminated fish ingestion) in wide variation: numbness, headache, eresthism, dizziness, cyanosis, prickling of lips, tongue and throat, nausea, vomiting, metallic taste, dryness of mouth, abdominal cramps and diarrhea. Sever case: reversal of temperature sensation, general

motor incoordination, paralysis and convulsion.

Fatality rate is low but recovery is slow and it takes several months.

More than 30,000 cases per year, most commonly reported disease in tropical and sub-tropical countries.

Ciguatera

Ciguatera causative dinoflagellate

Gambierdiscus toxicus

Benthic dinoflagellates living on seaweeds

Ciguatera is known endemic in Australia and tropical islands, but quite few in Western Pacific Asia. Recently Philippines has toxic fish.

Mechanism of ciguatera

From: BFAR Philippinestoxin producer herbivore

omnivore

carnivore patient

Countermeasures to ciguatera

Prohibition of marketing of possibly toxic fish species is only practical countermeasure to prevent poisoning.

Measurement of toxin amount is possible by HPLC, but standard toxins are difficult to obtain.

ELISA kits have been developed, but effectiveness is limited. Kits are very expensive.

Therefore, most countries do not have any countermeasure.




Mechanism of toxin accumulation

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Toxin

Excreted ToxinToxin producing microalgae

feeding microalgae

decomposing microalgae

excreting wastes

Gymnodinium catenatum

Alexandrium catenella

Pyrodinium bahamense

Human poisoning: Paralytic Shellfish Poisoning

PSP toxins produced from marine dinoflagellates of 3 genera, possibly the most geographically widespread marine biotoxins worldwide

in 1937, Sommer and colleagues established the association between toxic shellfish and blooms of the dinoflagellate Alexandrium catenella (then known as Gonyaulax catenella) along the California coast

toxins include the potent parent compound saxitoxin and approximately 20 variants of this basic structure which range considerably in toxicity

the regulatory limit for PSP toxins 80 µg STX equiv. 100 g-1 was established in the 1930s and is based on bioassays measuring toxic activity in mice

each year about 2000 cases of PSP are reported with 15 % mortality

levels of shellfish toxicity are dependent on the toxin profile and quota of the algae upon which they feed, the rate of uptake of algae by shellfish, capability of shellfish to transform, retain or depurate toxins


Saxitoxin

0 500 1000 1500 2000 2500Specific toxicity (MU/mol)*

STXneoSTXdcSTX

GTX1GTX2GTX3GTX4

dcGTX2dcGTX3

GTX5(B1)C1C2C3C4

HPLC analyzer determines each toxin component as its concentration (mM)

248322951274

2468892

158418031617

1872

15160

23933143

Toxicity(MU/g) can be calculated = Concentration x Specific toxicity for each toxin component


now realized that PSP toxins are more prevalent in food webs than previously thought

transfer of toxins to higher trophic levels has led to mortality events involving finfish, seabirds and marine mammals, e.g. whales having consumed toxic mackerel

shellfish typically serve as vectors of PSP toxins, but there are now many documented cases of shellfish mortalities

Paralytic shellfish poisoning （PSP）

Symptom is mainly paralysis of nerve system Tingling or burning sensation of lips, tongue and face starts

within ca. 30 min after ingestion. Paralysis of voluntary muscle extend to hands and legs. Patients keep conscious, but cannot move their body. Respiratory paralysis causes mortality within 12 hrs. High mortality (ca. 10%) Recover, if survival is over 12 hrs, within 48 hrs. No sequela.

No antidote. Facilitation of vomiting and urination is useful. Artificial respiration is the only a rescue.

Countermeasures to PSP

Prohibition of marketing of shellfish and other seafood possessing PSP toxins more than permitted level is the

only practical countermeasure to prevent poisoning.

Toxin amount is measured by mouse bioassay. Confirmation of toxins is by HPLC, but standard toxins are difficult to

obtain.

Most countries adopt 80 mg STX eq/100g edible body; Philippines takes 40 mg STX eq/100g edible body, because of monitoring difficulties.

ELISA kits have been developed, but effectiveness is still limited. Kits are very expensive.

Human poisoning:Diarrhetic Shellfish Poisoningfirst documented in 1976 [scallops in Japan]

very common form of poisoning[5000 cases in Spain in 1981]traditional ‘DSP complex’

okadaic [OA] and dinophysistoxins [DTXs] acid polyethers that inhibit protein phosphatase, and are the only toxins of the DSP complex with diarrheogenic effects in mammals

pectenotoxins [PTXs] are polyether-lactones which are hepatotoxic to mice by intraperitoneal injection

yessotoxins [YTXs] are disulphated polyethers that are cardiotoxic to mice, but are not associated with human poisonings

diarrhetic lipophilic toxins have been confirmed in at least 12 mixotrophic or heterotrophic species of Dinophysis

occasionally DSP toxins in shellfish have been associated with concentrations of Dinophysis as low as a few hundred cells l-1

Dinophysis species

Human poiosning: Diarrhetic Shellfish Poisonin

Diarrhetic shellfish poisoning （DSP）

Symptom always includes diarrhea. Vomiting and stomachache associates in more than 60% cases. Fever is rare. Symptom starts within 15 min to 4 hrs after ingestion. No mortality. Recover within 3 days. No sequela (?) cancer? Main toxin, okadaic acid, is cancer promoting agent.

Only bivalves are vector. No antidote.

HOOC OOOHH3C

O O

O OO

CH3

CH3

HOH

HO

H3C

CH3

OH H

31

35

2

7

Okadaic acid (OA)

Okadaic acid

one of DSP causative toxin no color, no smell, no taste, transparent heat stable, acid stable fat soluble

DSP occurrences and countermeasures

Occurrences in 1976 some hundreds cases in Japan by mussel in 1981-1983 several EU countries (Spain, France, Norway

etc.) had >8,000 cases by mussel Chile and New Zealand reported DSP recently in SEA very few report: Vietnam has several cases in Meretrix lyrata in Can Gio district from 1999 - 2002 causative food are only bivalves (mussel, scallop, clam, etc.)

Countermeasure prohibition of marketing of shellfish possessing DSP toxins

more than permitted level DSP toxin quantification by mouse assay in most countries. no monitoring in most SEA (only for foods for export)

Karenia brevis

Human poisoning: Neurotoxic Shellfish Poisoningcaused by brevetoxins produced by Karenia brevis [well known along the Gulf of Mexico, specifically the coast of Florida where it was first reported in 1844]

brevetoxins are lipid soluble cyclic polyethers which activate voltage-sensitive sodium channels causing sodium influx and nerve membrane depolarization [symptoms both neurological and gastrointestinal]

cell lysis allows toxins to be aerosolized by wind, wave and surf conditions causing respiratory and skin reactions in humans

Neurotoxic shellfish poisoning （NSP）

Symptom is neurological disorder such as tingling or burning sensation in mouth, which develops in the face, throat, and over the entire body, muscle paralysis and unusual temperature sensation within 1-2 hrs after ingestion. Patients had the sensation of being drunken. Diarrhea Patients keep conscious, but cannot move their body. Respiratory paralysis causes mortality within 12 hrs. No mortality Recover in a few days without any after effect. No antidote. At a red tide of K. brevis, another type of symptoms occur.

Neurotoxic shellfishpoisoning （NSP）

Characters of sick at red tide Responsible toxin is also brevetoxin Symptom appeared in people breathing wind containing volatile agents from dead red tide K. brevis cells is irritation of eyes and throats, accompanied by lacrimation and coughing. Mass mortality of fish and other marine organisms often occur at red tide bloom of K. brevis. Responsible agent is hemolytic compound, not brevetoxin.these are well described inhttp://www.floridamarine.org/features/default.asp?id=1018

Brevetoxin

Karenia brevis

Brevetoxin enhances inflow of Na+ inside nerve and muscle membrane.

Toxicity is strong. LD50 of brevetoxin B and

PbTX3 to mouse is 190 and 170 mg/kg, respectively.

Toxicity in shellfish is monitoredby mouse bioassay in USA,New Zealand, and Italy.

Amnesic shellfish poisoning （ASP）

Characters after ingestion of contaminated seafood

Poisoning cases was reported only in Canada with 108 patients including 3 mortality by softening of the brain. Symptom is vomiting, diarrhea, stomachache, headache, and diminution of appetite. In sever case, lost memory (amnesia), confusion, and lost of sense of balance and paralysis occur. In heavy case, patients lose conscious and die. Recover is slow. Amnesia is obvious. Mortality cases of birds and marine mammals were reported in USA, Argentine, and Mexico.

No antidote

domoic acid

N H

COOH

Me

MeCOOH

COOH3

53'

2

2' 1'

44'

5'

6

H2N COOH

COOH

Responsible toxins of ASP

N H

COOH

Me

COOH

2

36

kainic acid

Main toxin, domoic acid, is water soluble amino acid. Domoic acid and kainic acid compete with glutamic acid

to react to a receptor of nerve, and connect to it more than 10 times stronger than glutamic acid.

Glutamic acid is stimulant transmitter in the central nervous system. By domoic acid, glutamic acid cannot work.

Domic acid breaks memory center of brain irrecoverably.

glutamic acid

ASP causative organisms

The genus Pseudo-nitzschia has more than 33 species. 22 of them are non-toxic or not confirmed its toxin productivity. Pseudo-nitzschia spp.

Pseudo-nitzschia produces domoic acid only late-log to stationary growth phases

13 diatoms are confirmed its toxin productivity 11 Pseudo-nitzschia species

P. australisP. callianthaP. delicatissimaP. fraudulentaP. galaxiaeP. multiseriesP. multistriataP. pseudodelicatissimaP. pungensP. seriataP. turgidula

1 Nitaschia species: N. navis-varingica 1 Amphora species: A. coffaeiformis

has also caused intoxications in wildlife [sea lions, whales, sea otters, sea birds]

has however caused significant closures of fisheries, e.g. recreational razor clam harvesting

since 1987 monitoring has successfully prevented other human incidents of ASP

Human poisoning: Amnesic Shellfish Poisoning

Countermeasures to ASP

Prohibition of marketing of seafood possessing ASP toxins more than permitted level (20mgDA/kg edible part) is the only practical countermeasure to prevent poisoning.

At the time of poisoning occurrence in Canada, patients took 60-110 mg had mild symptom, but those 115 - 290 mg had serious poisoning.

Toxin amount is measured by LC (Liquid Chromatography).

ELISA kits have been developed for monitoring purposes.

Most of countries, except EU, North America, and Oceania, do not have ASP monitoring system.

Human poisoning: Azaspiracid Shellfish Poisoning

Protoperidinium crassipes identified as a potential producer of AZA, however the heterotrophic nature of this dinoflagellate requires consideration of its potential role as a vector rather than progenitor of these toxins

Protoperidinium crassipes

AZAs are marine polyether toxins also co-extracted with other lipophilic toxins, providing a single response in conventional mouse bioassays for DSP

caused human illness in 1995 in the Netherlands following ingestion of shellfish from Killary Harbour, Ireland [symptoms typical of DSP but DSP toxins were below regulatory levels]

Azaspiracid Shellfish Poisoning (AZP)

Protoperidinium crassipes

AZP toxicity in mussels is monitored by mouse bioassay or LC/MSin Europe.

Found in Netherlands in 1995 by mussels harvested in Ireland. So far reported only from mussels in Ireland water. Main symptoms such as nausea, vomiting, diarrhea and stomach cramp were similar to DSP, but responsible toxinwas different, neither OA nor DTXs. Causative organisms are dinoflagellates such as Protoperidinium crassipes.

Azadinium spinosum

AZP remains a rare illness and only five intoxication events reported to date [may be a high percentage of under reporting]

EU regulatory limit of AZAs 160 µg kg-1

Tillmann et al. (2009) recently identified Azadinium spinosumas the source of AZA

Human Health: Azaspiracid Shellfish Poisoning

Protoceratium reticulatum

Lingulodinium polyedrum

Human Health: Yessotoxins

Gonyaulax spinifera

YTXs have been associated with DSP as they are simultaneously extracted with DSP toxins and give positive results when tested with the mouse bioassay

recent evidence indicates that they should be excluded from the DSP toxins group as they do not cause diarrhea and date no human intoxication has been reported

yessotoxins [marine polyether toxins] known to be produced by 3 dinoflagellate species

YTXs have been found to be potent cytotoxins prompting the EU to establish a maximum permitted level of 1 mg YTX equiv. kg-1

Palytoxin

is a large, complex polyhydroxylated compound; one of the most potent toxins so far known

Human poisoning: Palytoxin

first isolated from the soft coral [Palythoa toxica] and subsequently from many other organisms including the benthic dinoflagellate, Ostreopsis siamensis

has caused mortalities of molluscs and echinoderms

cases of human deaths have been related to the consumption of crabs, sea urchins and fishOstreopsis siamensis

Microcystis aeruginosa

toxic cyanobacteria more common in freshwater, e.g. Microcystis aeruginosaproduces potent hepatotoxins called microcystins

WHO set an advisory limit of 1µg microcystin-LR equiv. l-1drinking water

toxic Microcystis blooms threaten wildlife and farm animals, human health, and recreational activities

Human poisoning: Hepatotoxicity

Poisoning Problems in Western Pacific

PSP: Serious 1980s and 1990s, but few cases after 2000; now recurrence problem causative species increases; area increasesDSP: Toxic dinoflagellates are detected, but no

monitoring on toxicity in shellfishASP: Toxic diatoms are detected, but no

monitoring on toxicity in shellfishNSP, AZP: No studies Ciguatera: 1997- Philippines and Hong Kong;

several studies on benthic dinoflagellates, but few on toxicity of fishes


1. Toxic by taking toxic organisms such as microalgae by feeding activity


2. Toxic by its own toxin production (either symbiotic bacteria or encode in gene)

example: Puffer,Blue-ringed octopus,Goby fish,Snail,Horseshoe crab, Xanthid rab…

Toxic animals

Some common marine puffer fish in SEA (Species name upper from left; Takifugu oblongus, Lagocephalus inermis, L. lunaris, middle from left; frL.

suezensis, Torquigener gloerfelti, T, brevipinnis, lower from left; Arothron hispidus, A. nigropunctatus, Canthigaster valentine)

Horseshoe crab Carcinoscopius rotundicauda

Goby fish Youngeichthys nebulosa

Blue-ringed octopusHapalochlaena lunulata

Marine toxic snails

Some toxic snails in Vietnam: From left, up to down:Charonia tritonis, Nassasius conoidalis, Oliva cericea, Polinices didyma, Nassasius catus*, Tutufa bufo, Tubo chysostomus, Tectus pyramis*:Causative species for human poisonings in VN

Chemical structure of Tetrodotoxin

Symptoms of poisoning•Main symptom of TTX poisoning is paralysis, whichis similar to PSP, which appears in 0.5 - 5 hoursafter eating. Death may occur within 1.5 - 8 hourswith lethal dose to human (50 kg) about 2 mg. Ifpatients survive for 8 hours, they will recoverwithout any residual longer effects.

Regulation•No regulation of TTX in toxic animals such aspuffers has been set in most countries. In Japan,10 MU is regarded as a limit of safe-consumptionlimit for puffer meat and skin (Kodama and Sato,2005).

・TTX and PSP occur in various but limited species in high concentration.

・A low level of TTX and PSP can be detected commonly in aquatic animals

・Causative organisms could be microorganisms.

Bacterial production of toxins

TTX: Yasumoto et al. (1986)Noguchi et al. (1986)

PSP: Kodama et al. (1988)

ProblemsToxin production is very lowBacteria do not always produce toxins

Kodama et.al. 2000

Characters of toxicitySpecies variationSeasonal variationGeographical variationIndividual variation (very wide in case of

puffer)Organ variationOccasionally (marine snails)

So, poisoning is unprediction, difficult to regulate/control

Numbers of poisoning cases, victims and death by eating puffer in Vietnam

Deaths by eating puffer is 42,9% of deaths by food poisoning Japanese cases are in ()

year case nos patients casualty1999 30 （20） 105 （34） 16 （2）

2000 27 （29） 129 （40） 29 （0）

2001 48 （31） 211 （52） 29 （3）

2002 49 （37） 210 （56） 29 （6）

2003 22 （38） 82 （50） 24 （3）

total 176 （155） 737 （232） 127 （14）

Importance:

Education to local peoples on scientific facts.

Documents

Toxin and toxic organisms causing food poisoningiocwestpac.org/files/upload_manual/Lecture 1.pdf · 2014-11-12 · Toxin and toxic organisms causing food poisoning Dao Viet Ha Head