Agricultural microbiology seminar on 12/09/2015. It deals with Scombroid as well as non-scombroid fish poisoning, along with cheese and other food intoxications. Epidemiology, pathophysiology, diagnosis, treatment and strategies to control are the aspects under consideration.

1. HISTAMINE POISONING
BY
BACTERIA
Debarshi Dasgupta
PALB-4137

3. SOMETHING as simple as storing fish in a colder area could have saved the lives
of Noelene Bischoff and her teen daughter Yvana.
Queensland authorities, who took over the autopsy from Indonesian investigators at the
request of the Bischoff family, have said early indications show scombroid food
poisoning, coupled with asthma suffered by the pair as well as mother Noelene's
migraine medication, may have caused their deaths.

5. ●Histamine poisoning is a food-borne chemical
intoxication resulting from the ingestion of foods that
contain unusually high levels of histamine.
Incubation period ranges from several minutes to a few
hours after ingestion of meal.
Centre for Disease Control(CDC) refers to it as :
●SCOMBROID FISH POISONING/HISTAMINE
FISH POISONING(in fish)
●HISTAMINE POISONING (in other food items
like Cheese,Sauerkraut,Ham etc.)

6. HISTAMINE
• Histamine, 4-(2-aminoethyl)imidazole , is a primary amine
arising from the decarboxylation of the amino acid, L-histidine.
• Tissues like liver and spleen contain endogenous histamine.
• Mast cells, basophils, entero-endocrine cells produce histamine.
• Mediated by receptors , effects include : cell proliferation and
differentiation, vasodilation, hematopoiesis, embryonic
development, wound healing, neurotransmission.
• Influences cognition, memory, vigilance and Circadian rhythms.

7. HAZARD IDENTIFICATION
Evidences of histamine being hazardous include:
1. Symptoms identical to those of IV histamine administration.
2. Efficient antihistamine therapy.
3. Presence of increased levels of histamine in the fish.
• Consuming spoiled fish containing histamine is more likely
to cause toxic effects than taking the same amount of pure
histamine by mouth.
• This has led to speculation that there are other „„scombroid
toxins‟‟ acting with histamine.

8. OCCURRENCE OF FISH POISONING
• Perhaps the most common type of food poisoning globally.
• Under-reporting of the disease is a worldwide problem.
• Many probable cases have been ignored as mild allergies, or
misdiagnosed.
• First described in 1799 in Great Britain.
• Re-emerged in medical consciousness when outbreaks in
Japan were noted in 1950‟s.
• USA,UK and Japan are the countries with most incidences.
• In 1973, Japan recorded the largest outbreak involving 2656
people.
• The fish mostly involved are Tuna, Mahi-mahi, Escolar,
Marlin and Salmon.
• Tuna and Mahi-mahi make up more than 80%of the
reported cases.

9. “SCOMBROID”: Associated with the fish in families Scombridae and
Scomberesocidae.
(a)Scombridae
Yellowfin tuna Thunnus albacares
Blackfin tuna T. atlanticus
Skipjack tuna Katsuwonas pelamis
Bullet tuna or bullet mackerel Auxis rochei
Atlantic bonito Sarda sarda
Atlantic mackerel Scomber scombrus
King mackerel Scomberomorus cavalla
(b)Scomberesocidae
Atlantic saury Scomberesox saurus
Pacific saury or mackerel pike Cololabis saira

10. SKIPJACK TUNA
BULLET TUNA ATLANTIC BONITO
ATLANTIC SAURY

11. NON-SCOMBROID FISH INVOLVED IN HFP:
(a)Pomatomidae
Bluefish Pomatomus saltatrix
(b)Coryphaenidae
Dolphin fish, dorado, or mahi-mahi Coryphaena hippurus
(c) Carangidae
Horse mackerel Trachurus trachurus / T. japonicus
Pacific amberjack Seriola colburni
(d) Clupeidae
Atlantic herring Clupea harengus harengus
Pacific sardine or pilchard Sardinops sagax
Golden sardine Sardinella aurita
(e)Engraulidae
European anchovy Engraulis encrasicolus

12. BLUEFISH
MAHI MAHI
HORSE MACKEREL ATLANTIC HERRING
ANCHOVY
PACIFIC
PILCHARD

13. OCCURRENCE OF CHEESE POISONING
• In Netherlands, in 1967, a person fell sick due to
consumption of Gouda cheese.
• In 1970‟s, cases were recorded in USA after consumption of
Swiss cheese.
• Gruyere cheese poisoning occurred in France.
• Cheshire cheese and Cheddar cheese poisoning have also
been reported in patients under Isoniazid therapy.

14. OTHER FOOD ITEMS PRONE TO POISONING:
Proteinaceous foods subjected to putrefaction and fermented
foods are particularly likely to contain large amounts of
histamine. Eg:-
• Sauerkraut ,wine, and fermented, dry sausage such as Italian
salami and pepperoni
• Chicken, Shellfish and Ham.
SALAMI
PEPPEROMI SHELLFISH

15. SYMPTOMOLOGY
Cutaneous
• Rash
• Urticaria
• Edema
• Localized inflammation
Gastrointestinal
• Nausea
• Vomiting
• Diarrhea
• Cramping
Hemodynamic
• Hypotension
Neurological
• Headache
• Palpitations
• Flushing
• Tingling
• Burning
• Itching

16. URTICARIA
DIARRHEA
FLUSHING ITCHING

17. FORMATION OF HISTAMINE
• Related to the enhanced histidine content of the fish, the
presence of histidine decarboxylase (HD) from bacteria.
• During spoilage, decarboxylases help in the formation of
other biogenic amines like:
a. Putrescine: from ornithine
b. Cadaverine: from lysine
c. Spermidine: from arginine
d. Spermine: from arginine
• Spoilage by bacteria are enhanced at higher temperatures
post-mortem.

19. • Proteolysis, either autolytic or bacterial, may play a role in
the release of free histidine from tissue proteins.
• (Milk does not contain large quantities of free histidine.)
• An alternate pathway, favoured by most bacteria,involves
glutamate as the ultimate product.
1. Histidine histidine ammonia lyase Urocanic acid
2. Urocanic acid α-ketoglutarate+ glutamate
Urocanic acid, like histamine, an imidazole compound derived
from histidine in spoiling fish, may be the „„missing factor‟‟ in
HFP.

20. METABOLISM OF HISTAMINE IN MAMMALS
Several routes:
1. Oxidative deamination by Diamine Oxidase (DAO) to form imidazole
acetaldehyde and imidazole acetic acid.
2. Methylation by histamine methyl transferase(HMT).
Oxidative deamination pathway is dominant in rats and guinea pigs.
Methylation pathway is prevalent in humans, mice, cats,pigs, hamsters.
• HMT-mainly in liver.
• DAO- GI tract.
• Monoamine Oxidase(MAO) is also important in histamine
metabolism
• MAO,DAO,HMT-inhibitors accentuate Histamine poisoning.

21. METABOLIC PATHWAYS OF HISTAMINE

22. MICROBIAL ASPECTS OF HISTAMINE POISONING
• Around 112 species of bacteria are known to possess Histidine
decarboxylase.
• Enterobacteriaceae, Clostridium sp., Lactobacillus sp. are widely
prevalent.
• Morganella morganii, Klebsiella pneumoniae and Hafnia alvei are
most prolific histamine producers when fish are maintained at 4°C or
more.
• Recently , Clostridium perfringens has been identified as a prolific
producer from skipjack tuna.
• For HFB: 2 media have been prepared:
1. Tuna fish infusion broth(TFIB): prepared from muscle tissue of raw
tuna.
2. Histidine-fortified trypticase soy broth: to maximize histamine
production.
3. MRS broth: For Lactobacilli

23. M. morganii K.pneumoniae
Lactobacillus sp.

24. According to temperature-growth relationships, the available data
suggests that there are no clear-cut distinctions between the temperature
groups………. But it‟s a continuum of values.
(A=Photobacterium phosphoreum; B= Vibrio sp.; C= Klebsiella, Morganella, Hafnia; D= if
present)

25. Lower limits for production of toxicologically significant
levels of histamine in tuna fish infusion broth(TFIB) for some
common HDB:
• 7°C- for Klebsiella pneumoniae
• 15°C- for Morganella morganii
• 30°C-for Hafnia alvei, Citrobacter freundii and
Escherichia coli.
Diversity of microflora in spoiling fish is varied, depends on
species, handling procedures, holding times and temperatures.
Character of microflora is influenced by the fish‟s feeding
habits, geographical location, season, temperature etc.
Indian preservatives like garlic,ginger, turmeric and black
pepper have been seen to have an inhibitory effect on the HDB
at concentrations 1%-5%.

26. Behling and Taylor(1982) divided HDB into 2 groups:
1. Those capable of producing
• >100mg Histamine/100ml TFIB
• in short incubation under 24 hours
• at temperature more than 15°C.
2. Those capable of producing
• <25mg Histamine/100ml TFIB
• in prolonged incubation more than 48 hours
• at temperature more than 30°C.

27. • Salt-tolerant lactobacilli may have been responsible for
histamine formation in the Gouda cheese implicated in a
histamine poisoning episode in the Netherlands.
• From swiss cheese some non-prolific histamine-producing
bacteria including Streptococcus faecium, S. mitis,
Lactobacillus bulgarius, L. plantarum, streptococci of the
viridans group, and propionibacteria, were isolated.
• Recently, a strain of L. buchneri from a sample of Swiss
cheese implicated in an outbreak of histamine poisoning, was
shown to be a prolific histamine producer.
• Further research will be needed to determine if additional
species of dairy-related bacteria are capable of significant
histamine production.

28. ANALYTICAL METHODS FOR DETECTION OF
HISTAMINE
• For detection of HDB, selective or differential media are used.
Histamine is usually extracted and measured commonly by
fluorometric assay.
• Various methods have been employed like:
1. Fluorometric assay
2. Thin-layer chromatographic method
3. High performance liquid chromatography
4. Capillary electrophoresis
5. Copper chelation method
6. Capillary zone electrophoresis, etc.
ELISA kits like ALERT provide good reproducibility. Enzyme
immunoassay kits like Histamarine kit is also increasing in popularity.
These are more sensitive, but expensive for routine studies.

29. POSSIBLE MECHANISMS OF TOXICITY
1. TOXICITY OF BIOGENIC AMINES:-
• By interaction with receptors on cell
membranes(H1,H2,H3,H4)
• But the dose-response relationship is not straightforward.
• Variable factors associated with the bacteria as well as the
host, are tough to quantify or disentangle.
• Normally , content of histamine 50mg/100g or more is
considered toxic.
• Histidine levels vary from 1g/kg in Herring to as much as
15g/kg in Tuna.

30. 2. HISTAMINE POTENTIATORS:
• Potentiators act to decrease the threshold dose of histamine
needed to cause adversity.
• Histamine-potentiator hypothesis states that
“Adsorption,metabolism and potency of one biogenic amine
might be modified in presence of a second amine”.
• Cadaverine and putrescine have been found to completely
inhibit DAO.
• Most potent inhibitors include tyramine(MAO inhibitor), β-
phenylethylamine(DAO,HMT inhibitor) and
tryptamine(DAO inhibitor).
• Pharmacological inhibitors are more potent. Include
cadaverine hydrochloride, tyramine hydrochloride, isoniazid
compounds.

31. 3. BARRIER-DISRUPTION HYPOTHESIS:
• “Potentiators might interfere with the protective actions of
intestinal mucin, which is known to bind histamine”
• Cadaverine exhibits a marked influence on the rate of
transport of C-14 labelled histamine and metabolites across
the gut wall in isolated gut sections of guinea pig.
4. ABSORPTION FROM MOUTH AND THROAT:
• Histamine maybe absorbed by mucous membranes of
mouth and throat, thus bypassing the digestive process that
destroys it

32. 5. RELEASE OF ENDOGENOUS (MAST CELL) HISTAMINE BY
SCOMBROID TOXIN:
• Mast cells contain a histamine-heparin complex.
• In 1972, Olley postulated “some other basic substance
produced during spoilage may release histamine from the
histamine –heparin complex.”
• Scombroid toxin is a mast cell degranulator.
• Imidazole compounds have been known to release
histamine from mast cells.
• Urocanic acid(imidazolic nature) has been found as a mast-
cell degranulator in human skin organ cultures. It may be
one of the “scombroid toxins” that scientists have been
searching for decades.

33. HISTAMINE LEVELS AND TOXIC DOSE
Threshold toxic dose for histamine in foods is not precisely
known and its difficult to determine.
Shalaby suggested the following guideline levels:
• <5mg/100g= safe for consumption
• 5-20mg/100g= possibly toxic
• 20-100mg/100g=probably toxic
• >100mg/100g=toxic and unsafe for consumption
FDA Guidelines for tuna,mahi-mahi and related fish:
• 500ppm(50mg/100g) as the toxicity level
• 50ppm(5mg/100g) as defect action level.

34. HUMAN FACTORS DETERMINING CLINICAL
RESPONSE:
1. VARIATION IN INDIVIDUAL SUSCEPTIBILITY:
Varies according to the amount of toxin ingested and variation
in individual susceptibility. Gradation of symptomatic
intensity.
2. INFLUENCE OF DIET:
• Geiger(1955) found that prior intake of seasoned spoiled
fish items and alcohol makes more adverse impact on the
system.
• Absorption is greater when taken with a meal like bread-
butter or milk etc.
• Potentiators from other foods like meat, cheese, fermented
foods like soy sauce and sauerkraut increase poisoning.
• Mixed seafood dishes may promote HFP.

35. 3. DISEASES AND AGE:
• Histidinaemia affected people lack HAL, so easily
poisoned.
• Histamine metabolism reduces with age.
4. INFLUENCE OF MEDICATION:
• HMT is inhibited by analogues of methylmethionine
such as adenosyl-homocysteine, anti-malarial drugs
and antagonists of histamine receptors.
• DAO inhibited by aminoguanidine.
• Isoniazid(anti-tuberculosis) is inhibitor of both MAO
and DAO.
• Antihistamines taken, may protect from HFP.

36. DIAGNOSIS
• Attention should be given to the type of fish ingested,
whether the fish was cooked or raw, whether these reactions
have occurred in the past, and the time frame between fish
ingestion and the onset of symptoms.
• An assessment of spoiled fish based on appearance and odor
alone does not aid in diagnosis. Instead, it is necessary to
test the histamine levels directly.
• In the allergy clinic, where in vitro testing for tissue levels of
histamine in fish is not available, skin prick testing (SPT)
can help diagnose HFP.

37. SKIN PRICK TEST

38. TREATMENT
• Antihistamines are the mainstay of treatment.
• For mild to moderate symptoms, effective oral H1 antagonists include
diphenhydramine, cetirizine, and chlorphenarimaine
• Cetirizine is preferred because it is less sedating.
• H2 blockers such as cimetidine, famotidine, or ranitidine can also be
added.
• If nausea is present, intravenous promathazine can be used and
intravenous fluids are indicated for diarrhea.
• Intravenous diphenhydramine and ranitidine or famotidine are the
drugs of choice in case intensity is serious.
• Intravenous fluids are indicated in treatment of hypotension.

40. FACTORS AFFECTING CHANCES OF OCCURRENCE
OF HISTAMINE POISONING
1. POST CATCHING CONTAMINATION
• At several levels: aboard the fishing vessel, at processing plant,
distribution system and at the consumer level.
• Subburaj et al.(1984)investigated fish market environment of
Mangalore.
2. TEMPERATURE ABUSE ON FISHING VESSELS
3. INADEQUATE CHILL-STORAGE TEMPERATURES
4. INADEQUATE FREEZING AND THAWING PROCEDURES
5. TEMPERATURE ABUSE IN PREPARING DRIED AND
SMOKED PRODUCTS.
6. POOR CANNING PROCEDURES
7. LOW-QUALITY FERMENTED PRODUCTS.
8. TEMPERATURE ABUSE OF RAW TUNA FOR SUSHI
MARKETS.

42. CONTROLLING SCOMBROTOXIN
FORMATION
Rapid chilling of scombrotoxin- forming fish
immediately after death is the most important
element in the strategy to control HFP,
especially for fish that are exposed to warm
waters of air, and for tunas which generate heat
in their tissues.

43. FDA, 1999

44. Time required to lower the internal temperatures of fish after
capture depends on:
1. The harvest method
2. The size of the fish
3. The chilling method.

45. Once chilled, the fish must be maintained at freezing
till consumption.
Exposures to temperature more than 40°F should be
minimised.
• This time limit depends on whether the fish had been
previously frozen properly, or heat-processed
sufficiently.

46. Heat processed fish are at low risk for formation of toxin(provided they
are handled in a proper manner that eliminates further contamination).
(FDA,1999)

47. HEALTH POLICY INITIATIVES
• In 1996, FDA implemented the HACCP Program, a set of rules that
governs time and temperature requirements at critical control points
along the entire supply chain.
• However, it is difficult to ensure that boats are adhering to HACCP
and the compliance rates were low.
• Several histamine fish poisoning outbreaks have been traced back to
fish caught on private boats.
• Healthcare providers must be made aware of this under-recognized
disease.
• Physicians should report histamine fish poisoning cases to local and
state health agencies for monitoring.

48. HAZARD ANALYSIS AND CRITICAL CONTROL POINTS(HACCP)
Aims to identify steps in the supply chain of fish where human intervention is needed to
control histamine poisoning: each concerned step is termed as a CCP.
LIKELY CCPs UNLIKELY CCPs
Receiving Continuous brief steps like filleting
Processing, such as: Processing brief steps such as:
1.Thawing 1. Date code stamping
2. Brining 2. Case packing
3. Smoking 3. Assembly for distribution
4. Drying Frozen product storage
5. Mixing (salad preparation)
Packaging
Final chilling
Storing raw material, in-process product
and finished product under refrigeration

49. QUESTIONS POSED FOR FURTHER RESEARCH
• What are the exact role of potentiators, urocanic acid and other biogenic
amines in the disease? How do these work, both singly and in combination?
• Can elevation in histamine be detected by faster and portable methods?
• Can microbes be commercially used for detoxification of histamine? If yes
then which ones can? And how can they be utilised?
• How true is the hypothesis that HDB arise from post-catching
contamination?
• How to properly differentiate it from allergy?
• How does greater histidine content of these fish help in their physiology?
• How to develop faster, more efficient control strategies in compliance with
HACCP to eliminate HFP?
So on and so forth…….

50. THANK
YOU