Lehane Histamine Scombroid Report

Histamine (Scombroid) Fish
Poisoning
a review in a risk-assessment framework
Leigh Lehane and June Olley
National Office of Animal and Plant Health
Canberra 1999
Histamine Fish Poisoning
ii
Commonwealth of Australia 1999
This work is copyright. It may be reproduced in whole or in part subject to the inclusion of an
acknowledgment of the source and no commercial usage or sale. Reproduction, for purposes other than
those indicated above, requires prior written permission from the Commonwealth available from
AusInfo. Requests and inquiries concerning reproduction and rights should be addressed to the
Manager, Legislative Services, AusInfo, GPO Box 1920, Canberra, ACT 2601.
Note:
To ensure timely publication, this report has not been subjected to either formal external peer review or
professional editing. It was prepared for publication by the Animal Health Science and Emergency
Management Branch, of the National Office of Animal and Plant Health, Department of Agriculture,
Fisheries and Forestry, Australia.
Revised 4/5/2000
Published by:
Animal Health Science and Emergency Management Branch
National Office of Animal and Plant Health
Agriculture, Fisheries and Forestry Australia
GPO Box 858
Canberra ACT 2601
Preferred way to cite this publication:
Lehane, L. and Olley, J* (1999) Histamine (Scombroid) Fish Poisoning: a review in a risk-assessment
framework. National Office of Animal and Plant Health, Canberra.
* Dr June Olley is an Honorary Research Associate in the School of Agricultural Science at the
University of Tasmania.
Acronyms used frequently in this report:
DAO, diamine oxidase or histaminase
DSP, diarrhetic shellfish poisons
HACCP, Hazard Analysis and Critical Control Point
HAL, L-histidine ammonia lysase, or histidase
HD, histidine decarboxylase
HDB, histidine decarboxylating bacteria
HFP, histamine fish poisoning
HMT, histamine methyl transferase
MAO, monoamine oxidase
PSP, paralytic shellfish poisons
iii
CONTENTS
SUMMARY 1
Introduction 1
Hazard identification 1
Doseresponse assessment 3
Exposure assessment 4
Risk characterisation 5
1. INTRODUCTION 7
2. HAZARD IDENTIFICATION 8
2.1 Definition of hazard identification 8
2.2 Production of histamine, other biogenic amines and imidazole
compounds in spoiling fish 9
2.3 Metabolism of histidine and histamine in mammals 11
2.4 Possible mechanisms of toxicity 13
2.4.1 Toxicity of histamine and other biogenic amines 13
2.4.2 Inhibition of histamine detoxification by histamine potentiators 15
2.4.2.1 Background 15
2.4.2.2 In vitro studies 16
2.4.2.3 In vivo studies 16
2.4.3 Barrier disruption hypothesis 18
2.4.4 Release of endogenous (mast cell) histamine by scombroid toxin(s) 19
2.4.5 Are paralytic and diarrhetic shellfish poisons involved? 22
2.4.6 Absorption of histamine from mouth and throat 22
2.5 Clinical characteristics and treatment 23
2.5.1 Clinical signs and symptoms 23
2.5.2 Diagnosis 24
2.5.3 Treatment 25
2.5.4 Clinical complications 25
2.6 Detection of histamine and other biogenic amines in fish 25
2.6.1 Detection of histamine-producing bacteria 25
2.6.2 Analysis of histamine 26
2.6.3 Analysis of other biogenic amines, and chemical quality index 28
2.6.4 Analysis of urocanic acid 30

3. DOSERESPONSE ASSESSMENT 31
3.1 Definition of doseresponse assessment 31
3.2 Incidence of histamine fish poisoning 31
3.2.1 Background 31
3.2.2 Africa 32
3.2.3 Asia 32
3.2.4 Australia 32
3.2.4.1 Outbreaks described by Smart (1992) 33
3.2.4.2 Cases described by Brown (1993) 34
3.2.5 Canada 34
3.2.6 Europe 34
3.2.7 New Zealand 36
3.2.8 United States 36

(DoseResponse Assessment continued over page)
iv
3.3 Fish characteristics that affect the clinical response 37
3.3.1 Species 37
3.3.2 Intrinsic potential for histamine and cadaverine accumulation 38
3.3.3 Seasonal and other variability 39
3.3.4 Parts of fish consumed 40
3.3.5 Nature and amount of bacterial contamination in spoiling fish 40
3.3.5.1 The common histamine-producing bacteria 40
3.3.5.2 Timetemperature relationships and histamine production 41
3.3.5.3 Studies on variability in species/strain composition of microflora 43
3.3.5.4 Species/strain variability in decarboxylase activity 44
3.3.5.5 Bacterial destruction of histamine 46
3.3.6 Histamine levels and toxic dose 46
3.4 Human factors that affect the clinical response 48
3.4.1 Variation in individual susceptibility 48
3.4.2 Influence of diet 49
3.4.3 Influence of medication 49
3.4.4 Disease states and age 50
3.5 Morbidity and mortality rates 50

4. EXPOSURE ASSESSMENT 51
4.1 Definition of exposure assessment 51
4.2 Factors affecting the probability of histamine fish poisoning occurring 51
4.2.1 Post-catching contamination 51
4.2.2 Temperature abuse on fishing vessels 52
4.2.3 Inadequate chill-storage procedures 53
4.2.4 Inadequate freezing and thawing procedures 54
4.2.5 Temperature abuse in the preparation of dried and/or smoked products 55
4.2.6 Poor canning procedures 57
4.2.7 Low-quality fermented products 57
4.2.8 Temperature abuse of raw tuna for the sashimi market 57
4.3 Histamine levels in fish products in Australia 58
4.3.1 Survey of histamine in canned tuna 58
4.3.2 Survey of biogenic amines in fish products in the ACT 58
4.3.3 Monitoring by the Australian Government Analytical Laboratories 59
4.3.4 Monitoring by industry 59
4.4 Amounts and types of fish consumed, and at-risk population groups 59
4.5 Future exposure trends 60
5. RISK CHARACTERISATION 61
5.1 Definition of risk characterisation 61
5.2 Nature and magnitude of risk 62
5.2.1 Impact on human health 62
5.2.2 Impact on fishing industries 62
5.3 Uncertainties and problem areas in risk characterisation 62
5.3.1 Defining histamine fish poisoning and elucidating its pathogenesis 62
5.3.2 Investigating and managing post-harvesting contamination 63
REFERENCES 66
Appendix 1: Acknowledgments 80
1
SUMMARY
Introduction
Histamine fish poisoning (HFP) is a foodborne chemical intoxication caused by the
consumption of spoiled, or bacterially contaminated, fish. Fish species associated with
HFP are harmless when caught. They may still have a normal appearance and odour
after they have become toxic. Spoiled fresh fish, and frozen and smoked fish and
canned fish products have all caused the disease.
HFP occurs worldwide. Recent reports in the literature have suggested that it is a
significant public health and safety concern. Its true incidence has probably been
underestimated, because of under-reporting and misdiagnosis owing to confusion with
symptoms of other illnesses, particularly food allergy.
Histamine, a physiological amine involved in allergic reactions, is the main toxin
involved in HFP, but HFP is not uncomplicated histamine poisoning. Although the
disease is generally associated with high levels of histamine (_50 mg/100g) in spoiled
fish, its pathogenesis has not been elucidated.
Scientific information on HFP is reviewed here in a risk-assessment framework in
order to arrive at an informed characterisation of risk. An attempt has been made to
address, as accurately as possible, the questions: What causes outbreaks of HFP?
What are the underlying factors contributing to outbreaks? and What are the
consequences of outbreaks?
Hazard identification
The involvement of histamine as the main hazard in HFP is supported by: symptoms
identical to those of intravenous histamine administration or allergic reaction; the
efficacy of antihistamine therapy; and the presence of increased levels of histamine in
spoiled fish that cause the syndrome.
Histamine production in fish is related to the histidine content of the fish, the presence
of bacterial histidine decarboxylase (HD), and environmental conditions. Bacterial
decarboxylase enzymes acting on free histidine and other amino acids in the fish
muscle form histamine and other biogenic amines. Fish of the family Scombridae,
notably tuna and mackerel, contain abundant amounts of histidine and are most
commonly implicated. However, many species, both scombroid and non-scombroid
(e.g. mahi-mahi, bluefish and sardines), have caused HFP so the term scombroid fish
poisoning is a misnomer.
The main bacteria responsible for histidine decarboxylation and HFP are members of
the family Enterobacteriaceae. Specific bacteria present in the marine environment or
introduced during food handling produce HD, which converts histidine to histamine,
particularly when fish are not kept chilled or frozen. Other bacteria in spoiling fish
muscle, again often members of the Enterobacteriaceae family, produce other
breakdown products such as biogenic amines putrescine from ornithine and
cadaverine from lysine.
2
Histamine consumed in spoiled fish is more toxic than an equal amount of histamine
taken orally in an aqueous solution. It has been proposed that other biogenic amines
produced in spoiled fish may potentiate the action of histamine by inhibiting the
enzymes diamine oxidase (DAO, or histaminase) and histamine methyl transferase
(HMT). These two enzymes normally detoxify histamine in the intestinal tract and
prevent the absorption of unmetabolised histamine into the circulation. Putrescine and
cadaverine have been identified as histamine potentiators. However, results of
experiments to support the potentiator hypothesis are not totally convincing. Another
possible mechanism of potentiation of histamine by other biogenic amines is the
barrier disruption hypothesis, which suggests that potentiators might interfere with
the protective actions of intestinal mucin, which binds histamine.
Still more hypotheses have been proposed in an attempt to explain the pathogenesis of
HFP. Some scientists have suggested that a scombroid toxin (or toxins) in
scombrotoxic fish causes the release of endogenous histamine from mast cells, which
augments the exogenous histamine consumed in spoiled fish in causing symptoms of
toxicity. Others have proposed that paralytic and/or diarrhetic shellfish poisons are
responsible. Yet another hypothesis, long discarded, is that histamine in a fish
substrate may be better absorbed from the mouth or throat than pure histamine in
solution.
Thus, the pathogenesis of HFP remains obscure. After sifting the evidence, the present
authors believe that endogenous release of histamine from mast cells probably does
occur in HFP, at least in some cases. We suggest that urocanic acid may be the
missing factor (scombroid toxin) in HFP for the following reasons. In spoiling fish,
histidine may be metabolised to histamine, or to urocanic acid and glutamate by an
alternative metabolic pathway. The first step in the alternative pathway is the loss of
ammonia from histidine by the action of L-histidine ammonia lysase (HAL, or
histidase). HAL has a wide distribution among bacteria and, unlike HD, is also found
naturally in fish muscle. Bacteria also produce urocanase, but HAL activity is greater
than that of urocanase and urocanic acid accumulates with histamine in spoiling fish.
Most importantly, urocanic acid has recently been recognised as a mast cell
degranulator.
Histamine is catabolised by several routes in the human body. The main catabolic
pathways involve the enzymes DAO and HMT. HMT activity in particular is
widespread in many body tissues and imidazole histamine metabolites are excreted in
the urine.
Histamine exerts its toxicity by interacting with histamine receptors (H
1
, H
2
and H
3
)
on cellular membranes. The most common symptoms of histamine poisoning are
cardiovascular flushing, urticaria, hypotension and headache. Other symptoms are
gastrointestinal abdominal cramps, diarrhoea and vomiting and neurological
pain and itching associated with urticarial lesions. However, HFP is usually a mild
disease of quick onset (several minutes) and short duration (about 8 h). It responds
well to antihistamine treatment.
3
Numerous tests are available for detecting histidine decarboxylating bacteria (HDB)
and histamine. Problems associated with histamine detection in fish as a control or
diagnostic measure for HFP include: the lack of uniform distribution of histamine in
toxic fish; the large numbers of methods available and the lack of standardisation
between countries; and the fact that histamine is not the only factor involved in the
pathogenesis of HFP. High-performance liquid chromatography (HPLC) and capillary
electrophoresis are often used, and rapid enzyme-linked immunosorbent assay
(ELISA) test kits are also available. Methods including HPLC are used for detecting
other biogenic amines and urocanic acid.
Doseresponse assessment

Since 1970, most reports of HFP have come from Japan, the US and Great Britain.
Outbreaks have been reported less frequently in various other countries, including
Australia and New Zealand. There are only two reports of HFP in Australia in the
scientific literature. Juvenile Western Australian salmon caught in South Australian
waters were responsible for two outbreaks, affecting a total of seven people; and two
people were affected by eating a tuna meal at a restaurant in Brisbane. The true
incidence of HFP in Australia is unknown.
Scombroid fish that cause HFP include mackerel (Scomber spp.), tuna (Thunnus spp.),
saury (Cololabis saira) and bonito (Sarda app.) (scombroid fish). Non-scombroid fish
that cause HFP include mahi-mahi or dolphin fish (Coryphaena spp.), sardines
(Sardinella spp.), pilchards (Sardina pilchardus), anchovies (Engraulis spp.), herring
(Clupea spp.), marlin (Makaira spp.) and tailor or bluefish (Pomatomus spp.). Other
non-scombroid species, Western Australian salmon (Arripis truttaceus), sockeye
salmon (Oncorhynchus nerka) and Cape yellowtail (Seriola lalandii), have also been
implicated.
Since free histidine in fish muscle is the substrate for microbial decarboxylation to
produce histamine, species difference in free histidine content has a large effect on the
potential hazards of poor handling and storage practices. Levels of ornithine (to
produce putrescine) and lysine (to produce cadaverine) may also be important.
Histamine concentrations in fish tend to be greater adjacent to the gills or intestines.
Spoilage and ammonia and biogenic amine production are enhanced at elevated
temperatures, with histamine production by bacteria such as Morganella morganii,
Klebsiella pneumoniae and Hafnia alvei being optimal at 30
o
C. Once a large bacterial
population has been established, residual enzyme activity continues slowly at
refrigeration temperatures (05
o
C), although bacterial growth ceases. Histamine is
also produced, but to a lesser extent, by bacteria that can grow at refrigeration
temperatures (e.g Vibrio spp., Photobacterium spp.).
4
It is not possible to predict accurately the effect of temperature abuse on histamine
formation. Different bacterial species have different temperatures for maximum
growth, and the histamine concentration in any part of a fish represents that produced
by a population of bacteria with capacities for histamine production and/or destruction
ranging from high to zero. However, as spoilage progresses, the proportion of HDB in
the microbial population increases. In addition, quantification of bacterial
contamination is not meaningful. Subsequent heat processing can destroy bacterial
contaminants and even HD activity, but has little or no effect on histamine levels.
Although histamine is not solely responsible for HFP, levels of histamine in suspect
fish serve as an important indicator of bacterial contamination, and many countries
have set guidelines for maximum permitted levels. The current level for histamine in
fish in the Australian Food Standards Code is 100 mg/kg. However, concentrations of
histamine within a fish are extremely variable, as is the threshold toxic dose.
If we regard 10 mg histamine/100 g of fish as the highest level that can be consumed
safely by most people, this must be related to the amount of fish eaten and the weight
of the person to calculate a likely safe dose. If a 60-kg person eats, say, 300 g (wet
weight) of this fish, this dose would be 0.5 mg/kg bodyweight. Such a calculation is of
limited value, however, considering the variable nature of HFP and the lack of
understanding of its pathogenesis.
The severity of the clinical response depends on the amount of toxin(s) ingested and
the variation in individual susceptibility. In some outbreaks the morbidity rate may
reach 100%. There is large variation in individual susceptibility. Certain dietary
components and medications such as isoniazid, aminoguanidine and some
antihistaminic drugs increase susceptibility. Disease states, such as allergies and
mastocytosis, may also affect the clinical manifestation of HFP.
Exposure assessment
Post-catching contamination with HDB may occur aboard the fishing vessel, at the
processing plant, in the distribution system (fresh and frozen fish), and at the level of
the user, for example in a restaurant. The key to the reduction of histamine production
is the rapid cooling of the fish after catching.

As most histamine-producing bacteria require temperatures >15
o
C for growth, low-
temperature storage (<10C) effectively controls their growth. However, some
bacteria can produce smaller amounts of histamine in fish stored at temperatures
between 0 and 10C. Regardless of the species involved, bacteria must grow to a large
enough population for significant production of histamine to occur.

If fish are subject to elevated temperatures, even for short periods, a large microbial
population is established. During subsequent refrigeration, although bacterial growth
ceases, residual enzyme activity continues slowly and histamine (and putrescine and
cadaverine) levels continue to increase. If the fish are then hot smoked or canned, the
heat will destroy the residual microflora and HD, but not histamine.
5
Indications that HFP is not common in Australia are supported by results of limited
surveys and routine monitoring. Of 104 cans of tuna screened in Melbourne retail
markets in 1987 (45 from Australia and 59 from Asian countries), 101/104 cans had
<5 mg/100 g histamine, and 3/104 had 510 mg/100 g. In a recent ACT Government
survey of biogenic amines in seafood and seafood products, only 1/64 samples, dried
fish from Asia, failed to comply with the Australian Food Standards Code level of
100 mg/kg histamine. However, two dried fish samples contained >100 mg/kg
putrescine, and two contained >100 mg/kg cadaverine.

Ongoing random monitoring of imports and domestic product by the Australian
Government Analytical Laboratories (AGAL) has revealed only a small percentage of
samples with >100 mg/kg histamine. The only remaining tuna cannery in Australia,
Port Lincoln Tuna Processors Pty Ltd, South Australia, monitors histamine levels in
all batches of fish entering the cannery, as well as in finished product, to ensure that
strict food safety standards are maintained.
In developed countries where HFP still occurs quite frequently, such as the United
States and Japan, most outbreaks are the result of consumption of fish caught by
recreational fishers, with insufficient knowledge of the problem and without proper
chilling facilities on fishing boats.
Although not reported to any extent, HFP is probably still common in developing
countries, where fish preserved by traditional methods is an important part of the diet.
The problem would be expected to be greater in tropical countries, where high
ambient temperatures promote the growth of the most active HDB.
Great progress has been made in ensuring the quality of fish products despite the huge
expansion in trade in recent years. This is the result of the introduction of international
standards in food hygiene and the application of risk analysis and Hazard Analysis and
Critical Control Point (HACCP) principles. Although incidents of HFP caused by high
levels of histamine in canned tuna have occurred all over the world, improvements in
handling and processing associated with the establishment of quality control
procedures are now widespread and taking effect.
Risk characterisation
HFP does not have a major impact on human health in Australia, partly because fish
does not form a large part of the diet of most Australians. However, the disease is
important from the food safety aspect and it is possible that toxic products,
particularly imports, will escape the random monitoring safety net from time to time.
Consumers are becoming more demanding and litigation following food poisoning
incidents is becoming more common. Producers, distributors and restaurants will
increasingly be held liable for the quality of the products they handle and sell.
If a major outbreak of HFP were to occur in Australia, as has happened in Japan and
the United States, resulting media attention would affect fish consumption and have a
negative impact on the marketing of seafood. An outbreak in another country caused
by Australian exports would seriously affect trade. Although such events are
becoming increasingly less likely because of the widespread adoption of HACCP
6
analysis and quality assurance, constant monitoring is necessary to allow for factors
such as equipment failure, human error or negligence.
Other activities could also place Australia in a better position to characterise the risk
of HFP and prevent or rapidly manage an outbreak should it occur. The pathogenesis
of HFP requires elucidation. More research is needed to determine the threshold of
toxic dose for histamine and the role that potentiators or other toxins play in causing
toxicity. The possible role of urocanic acid in HFP, as a potential mast cell
degranulator, should be investigated, initially in laboratory animals. There is also a
need to better assess the actual incidence of fish allergy and to estimate what
percentage of cases diagnosed as fish allergy represent misdiagnosis of HFP.
There is a need for global standardisation of histamine detection methods, and
laboratory accreditation and proficiency testing, if histamine is to remain the main
indicator of microbial spoilage in histidine-containing fish. From an environmental
health perspective, a rapid and cheap assay for detecting histamine in fish would be of
value if made available to the public and, in particular, to recreational fishers.
However, monitoring fish histamine levels alone may not always ensure protection
from HFP. There may be advantage in the simultaneous detection of other biogenic
amines, such as cadaverine and putrescine. The detection of histamine, cadaverine and
putrescine can be achieved satisfactorily by the use of two-dimensional TLC or
HPLC, but this is neither rapid nor cheap. Urocanic acid may be a useful alternative to
histamine as a spoilage index in scombroid and other fish that are rich in endogenous
histidine, and may be linked to the potential of fish to cause HFP.
Reporting of suspected cases of HFP to local food authorities should lead to removal
of contaminated fish from the marketplace and prevention of additional cases.
Mechanisms should be put in place, where these are not present already, to allow
efficient and complete traceback of incriminated fish to point of origin, in order to
rectify problems leading to spoilage. In addition, there needs to be education of
recreational fishers and the public about the need for good refrigeration and hygiene to
minimise the possible hazards of consuming fish.
7
1. INTRODUCTION
Scombroid fish poisoning, or more correctly histamine fish poisoning (HFP), is a
foodborne chemical intoxication caused by eating spoiled, or bacterially
contaminated, fish. Fish species associated with HFP are harmless when fresh, and
after they have become toxic they may still have a normal appearance and odour
(Sapin-Jaloustre and Sapin-Jaloustre 1957). No available method of preparation,
including freezing, canning and smoking, will destroy the causative toxin(s) (Etkind et
al 1987).
Histamine which is the main toxin involved in HFP is also a naturally occurring
substance in mammalian physiology. It is contained in mast cells and basophils, and
its powerful biological effects are usually seen only when it is released in large
amounts in the course of allergic and other reactions. Histamine exerts its effects by
binding to receptors on cellular membranes in the respiratory, cardiovascular,
gastrointestinal and haematological/immunological systems and the skin (Cavanah
and Casale 1993). The pathogenesis of HFP has not been elucidated. The disease is
not uncomplicated histamine poisoning, but is generally associated with high levels of
histamine (_50 mg/100g) in spoiled fish.
Although it is a common form of fish poisoning, many incidents of HFP go
unreported because of the mildness of the disease, lack of required reporting, and
misdiagnosis. Symptoms may be confused with those of other illnesses, particularly
'Salmonella infection' (Russell and Maretic 1986) and food allergy (Taylor 1985;
Taylor et al 1989; Institute of Medicine 1991; Snchez-Guerrero et al 1997; Kerr and
Parke 1998). The syndrome is that of histamine toxicity, but there is individual
variation in susceptibility, and clinical signs are more severe in people taking
medications such as isoniazid, which inhibit enzymes responsible for normal
histamine-detoxification reactions in the intestine (Stratton et al 1991).
HFP is a significant public health and safety concern (Sanchez-Guerrero et al 1997;
Wu et al 1997; Fletcher et al 1998) and a trade issue (Anon. 1998a). The earliest
record of the disease was in 1828 (Henderson 1830). Since then, it has been described
in many countries, including Australia and New Zealand (Taylor 1985), and is now
the most prevalent form of seafood-borne disease in the United States (Lipp and Rose
1997). The worldwide network for harvesting, processing and distributing fish
products has made HFP a global problem. Because the toxic condition is a
consequence of improper handling or storage of fish and there are effective testing
methods to identify likely toxic fish, control and prevention are possible (Institute of
Medicine 1991).
Scientific information on HFP is reviewed here in a risk-assessment framework
(Kindred 1996; Buchanan et al 1998) in order to arrive at an informed characterisation
of risk. An attempt has been made to address, as accurately as possible, the questions:
'What causes outbreaks of HFP?', 'What are the underlying factors contributing to
outbreaks?' and 'What are the consequences of outbreaks?'
Risk assessment forms the scientific foundation for risk analysis, which comprises
risk assessment, risk management and risk communication. The purpose of risk
8
analysis is to foster informed management decision making. The risk assessment
presented here is intended for use in a subsequent study to weigh policy alternatives
and, if required, to select appropriate control options, including risk communication.
2. HAZARD IDENTIFICATION
2.1 Definition of hazard identification

Kindred (1996) defined hazard identification as The determination of whether a
particular chemical is or is not causally linked to particular health effects. Topics
discussed under hazard identification may include:

sources of the hazard;
the frequency with which it may occur in food, and in what amounts;
the usual food vehicle(s);
the mechanism of toxicity;
the pathogenesis, clinical signs, pathology and diagnosis of the disease the hazard
causes; and
methods of detection of the hazard.

The involvement of histamine as the main hazard in scombroid poisoning or HFP is
supported by:

symptoms identical to those of intravenous histamine administration or allergic
reaction;
the efficacy of antihistamine therapy; and
the presence of increased levels of histamine in spoiled fish that cause the
syndrome.
In 1991, Morrow et al claimed that there was definitive evidence that histamine is the
toxic agent in HFP after analysing the urine of poisoned subjects. They found that
urinary levels of histamine in patients with HFP were higher than those of subjects
who had been injected intravenously with histamine to produce toxic symptoms.

However, HFP is not simply histamine poisoning. Consuming spoiled fish containing
histamine is more likely to cause toxic effects than taking the same amount of pure
histamine by mouth. Pure histamine taken orally is substantially metabolised in
crossing the intestinal wall or in the liver, and produces only mild symptoms at high
doses (Taylor et al 1984). Thus, while elevated blood histamine levels are
undoubtedly characteristic of HFP, there is uncertainty about how these occur. The
pathogenesis of the disease is complicated and there is speculation about the identity
of other possible scombroid toxins acting in concert with histamine.

Taylor (1986) said that HFP appears to be caused by exogenous histamine (i.e. from
spoiled fish), potentiated by other biogenic amines and possibly other substances.
Clifford et al (1991) and Ijomah et al (1991), on the other hand, postulated that a
toxin or toxins other than histamine in the spoiled fish cause release of endogenous
histamine from mast cells in the human body. The indication that there may be
9
multiple toxins, perhaps acting synergistically, may account for the considerable
variability of clinical signs and symptoms encountered.

2.2 Production of histamine, other biogenic amines and imidazole compounds
in spoiling fish

Histamine production in fish is related to the histidine content of the fish, the presence
of bacterial histidine decarboxylase (HD), and environmental conditions (Ienistea
1973). During spoilage, certain bacteria produce decarboxylase enzymes, which act on
free histidine and other amino acids in the fish muscle to form histamine and other
biogenic amines. Chemically, histamine (from histidine), putrescine (from ornithine),
cadaverine (from lysine), and spermidine and spermine (from arginine), which are
produced post-mortem in fish muscle, are low-molecular-weight, aliphatic, alicyclic
or heterocyclic organic bases (Eitenmiller and De Souza 1984; Rawles et al 1996).

Fish species belonging to the families Scombridae (e.g. tuna and mackerel) and
Scomberesocidae (e.g. saury) are most commonly associated with HFP, but non-
scombroid fish, such as mahi-mahi, sardines, pilchards, anchovies, herring, marlin and
bluefish can also be involved (Taylor 1986). These fish species are characterised by
having relatively high levels of histidine in their flesh (Taylor 1986; Institute of
Medicine 1991). Histidine levels vary from 1 g/kg in herring to as much as 15 g/kg in
tuna (Ijomah et al 1992). Frank et al (1981) found that fresh (skipjack) tuna
(Katsuwonus pelamis) contained negligible quantities of histamine, usually
<0.1 mg/100 g.

Histamine can be produced rapidly by bacterial decarboxylases in scombroid and
other fish that have relatively high free histidine levels in their muscles when alive
(Love 1980), before post-mortem proteolysis liberates additional histidine from
muscle protein. This explains why toxic levels of histamine are generally limited to
the red meat of free-swimming species (Arnold and Brown 1978) and why histamine
can reach high concentrations without the formation of organoleptic (sensory)
spoilage indicators (Sapin-Jaloustre and Sapin-Jaloustre 1957). After investigating
HD production by Morganella morganii in mackerel, Eitenmiller et al (1982)
concluded that the ready availability of free histidine in the muscle to act as both an
inducer and substrate makes scombroid fish muscle an ideal environment for
histamine formation.

The main bacteria responsible for histidine decarboxylation and HFP are members of
the family Enterobacteriaceae (Frank et al 1985; Taylor and Sumner 1986).
Endogenous production of decarboxylase enzymes is insignificant when compared
with the exogenous (bacterial) pathway (Rawles et al 1996). Spoilage, ammonia
production and biogenic amine production are enhanced at elevated storage
temperatures, with histamine production being optimal at around 30
o
C (Arnold et al
1980). Once a large bacterial population has been established, residual enzyme
activity continues slowly at refrigeration temperatures (05
o
C), although bacterial
growth ceases (Institute of Medicine 1991). Histamine is also produced, but to a lesser
extent, by bacteria that can grow at refrigeration temperatures (Okuzumi et al 1981).

10
Only free histidine can be decarboxylated (Arnold and Brown 1978). Geiger (1944b)
observed that the ability of Escherichia coli to decarboxylate histidine was highly
specific, since histamine formation was inhibited by acylation of the amino group of
histidine. He later studied histamine formation with a Clostridium perfringens enzyme
preparation, a strain of E. coli and a strain of a marine bacterium. All were able to
form histamine from free L-histidine, but none were able to form histamine from
aspartyl-histidine or histidyl-histidine. Geiger (1944b) concluded that peptide linkages
to either the -COOH or -NH
2
groups of histidine prevented bacterial decarboxylation.

The decarboxylation of histidine to form histamine is only one of two routes of
histidine metabolism, and the occurrence of this pathway in fish spoilage is quite
limited. According to Baranowski (1985), the pathway favoured by most bacteria is a
catabolic one in which glutamate is the ultimate product formed. The first step in this
pathway is the loss of ammonia from histidine by the action of L-histidine ammonia
lysase (HAL), or histidase, resulting in the formation of urocanic acid (Fig. 1 below).

Figure 1. Degradation of histidine by HAL to form urocanic acid and ammonia. (From
White et al 1973.)

HAL has a wide distribution among bacteria (Shibatani et al 1974; Baranowski 1985)
and, unlike HD, is also found as an endogenous component of fish muscle (Kawai and
Sukaguchi 1968; Mackie and Fernndez-Salguro 1977). Some bacteria also possess
urocanase, which catabolises urocanic acid, but Shibatani et al (1974) found that no
strain of 106 tested had urocanase activity equal to its HAL activity. Urocanic acid
was found at a much higher concentration than histamine (4.74 versus
0.19 mg/100 mL) in mackerel after 18 days' storage at 0
o
C (Mackie and Fernndez-
Salguro 1977; Fernndez-Salguro and Mackie 1979), although in this experiment it
was attributed to endogenous rather than bacterial HAL.
Shewan (1955) investigated the distribution of free bases and amino acids in muscle
from fresh and spoiled mackerel by fractionation by displacement chromatography on
ion-exchange resins of aqueousalcoholic extracts. Following spoilage, he found large
amounts of histamine and moderate amounts of an unidentified (probably imidazole)
component (compound 8) not found in fresh muscle. Later, Hughes (1959)
11
demonstrated a major imidazole spoilage product (compound X) from spoiled herring
by paper chromatography, which gave cherry red colour with Paulys reagent,
suggesting the imidazole portion of the molecule was still intact. However, his efforts
to identify compound X as urocanic acid (imidazolylacrylic acid),
imidazolylcarboxylic acid or imidazolylacetic acid by comparison with authentic
samples (of unspecified origin) were unsuccessful.

2.3 Metabolism of histidine and histamine in mammals

Humans metabolise histidine to urocanic acid through the activity of HAL, to form
glutamate and then -ketoglutarate, which enters the citric acid cycle (White et al
1973; Furuta et al 1996), or to histamine through the activity of HD (Stryer 1981).

Histamine can be catabolised by several routes (Fig. 2). It can be oxidatively
deaminated by diamine oxidase (DAO, or histaminase) to imidazole acetaldehyde and
imidazoleacetic acid, methylated by histamine methyl transferase (HMT) to form
methylhistamine, or its side chain can be methylated or acetylated (Rice et al 1976;
Taylor 1986).

Figure 2. Histamine catabolic routes. (From Taylor 1986.)

12
The fact that oral histamine alone is not toxic to adults except at doses 100 mg
appears to be due mainly to the presence of DAO and HMT in the intestinal tract,
which detoxify histamine (references cited by Taylor 1986). Histamine is also
converted to inactive acetylhistamine in the intestine, presumably by bacterial enzyme
(references cited by Taylor 1986). All these enzymes decrease the amount of
unmetabolised histamine available for absorption into the circulation (Granerus 1968;
Taylor and Lieber 1979; Lyons et al 1983; Hui and Taylor 1985; Taylor 1986).

The relative importance of the DAO and HMT pathways varies among species. The
oxidative deamination pathway predominates in rats and guinea pigs, and the
methylation pathway is of prime importance in humans, mice, cats, pigs and hamsters
(Taylor 1986).

When [
14
C]-histamine was administered orally to humans, 6880% of the radioactive
dose was recovered in the urine (Sjaastad and Sjaastad 1974). Some histamine
remained unchanged in the faeces, and additional amounts were catabolised by
intestinal bacteria and radioactivity was exhaled as
14
CO
2
from the lungs. Hesterberg
et al (1984) demonstrated that HMT activity was widespread in human tissues, with
the order of activity being liver >> colon >spleen > lung > small intestine > stomach.
By contrast, DAO was mainly localised in the intestine (Hesterberg et al 1984).
Helander et al (1965) had shown earlier that the human kidney possessed considerable
capacity for removing histamine from the blood. When they infused healthy
individuals with histamine intravenously, a large proportion was methylated by the
kidney and excreted in the urine, while a smaller proportion was excreted unchanged
in the urine.

HMT is very selective for histamine, and requires S-adenosylmethionine as a methyl
donor. HMT is inhibited by analogues of methylmethionine, such as adenosyl-
homocysteine, antimalarial drugs and numerous agonists and antagonists of histamine
receptors. HMT is also subject to substrate inhibition by high concentrations of
histamine. The type of inhibition (competitive, uncompetitive, non-competitive)
varies depending on the inhibitor. (References cited by Taylor 1986.)

DAO oxidises other diamines, such as putrescine, as well as histamine. Many
inhibitors of DAO have been identified, such as aminoguanidine and some
antihistaminic drugs. DAO is also subject to substrate inhibition when certain
diamines including histamine are used as substrates. A number of foodborne
substances are inhibitors of DAO, including carnosine, thiamine, cadaverine and
tyramine. Monoamine oxidase (MAO) is also important in histamine metabolism.
(References cited by Taylor 1986.)

13
2.4 Possible mechanisms of toxicity

2.4.1 Toxicity of histamine and other biogenic amines

In the body, histamine exerts its toxicity by interacting with receptors on cellular
membranes. There are three types of histamine receptors, H
1
, H
2
and H
3
(Cavanah and
Casale 1993). The most common symptoms of histamine poisoning result from the
action of histamine on the cardiovascular system, which involves H
1
and H
2
receptors.
Histamine causes dilatation of peripheral blood vessels, capillaries and arteries,
causing urticaria, hypotension, flushing and headache. Histamine-induced contraction
of intestinal smooth muscle, mediated by H
1
receptors, causes abdominal cramps,
diarrhoea and vomiting. Pain and itching associated with the urticarial lesions may be
due to sensory and motor neuron stimulation, which is also mediated by H
1
receptors
(Taylor 1986).

There is compelling evidence to implicate histamine as the causative agent in HFP
high levels of histamine have been found in food samples implicated in outbreaks, the
symptoms noted in the patients are consistent with histamine poisoning, and
antihistamines are effective in counteracting the symptoms. However, there is not a
straightforward doseresponse relationship. Spoiled fish containing histamine tends to
be more toxic than the equivalent amount of pure histamine dosed orally. Studies on
HFP have been confused by interacting variables affecting both fish samples and the
consumer and, according to Ijomah et al (1992), most investigations have been limited
in their ability to disentangle these. Potentially confusing factors are listed in the table
below.

Potentially confusing factors in HFP

Samples Consumers
Variable histamine levels in sample
Microbial toxins
Other toxins/contaminants/
metabolites
Wrong sample analysed

Wrong diagnosis
Bodyweight differences
Individual variation
Sex differences in metabolism
Concomitant medication
Idiosyncratic intolerance
True allergy

Weiss et al (1932) found that 180 mg histamine base (given as 500 mg histamine
phosphate) administered orally was without noticeable effect in humans, while 7 g
administered intravenously caused vasodilatation and increased heart rate. Granerus
(1968) gave humans up to 67.5 mg histamine orally without toxic effect. Motil and
Scrimshaw (1979) found that administration of 100180 mg histamine orally mixed in
grapefruit juice (containing citric acid) or in 100 g high-quality tuna caused
characteristic symptoms of mild histamine poisoning (mild-to-severe headache and
obvious flushing) in some people (1/4 and 4/8 respectively). These doses represented
1.63 mg/kg bodyweight (bw), assuming an average weight of 60 kg. Subsequently,
Clifford et al (1989) gave volunteers 50 g of fresh mackerel to which 300 mg
histamine had been added (a dose of about 5 mg/kg bw), and recorded only mild
symptoms of histamine poisoning (oral tingling, headache and flushing in some
14
subjects). However, 50 g of spoiled mackerel with 300 mg added histamine was no
stronger in its effect.

Theories have been put forward to help explain why fish that cause histamine
poisoning often do not contain levels of histamine considered high enough to be toxic
orally. Some scientists maintain that, in HFP, factors (potentiators) act to make
exogenous histamine more toxic, possibly by increasing its absorption or decreasing
its detoxification in the intestinal tract or elsewhere in the body, such as the liver
(Taylor and Lieber 1979; Lyons et al 1983; Hui and Taylor 1985). Others believe that
another toxin or toxins might act to liberate endogenous histamine, which contributes
to histamine toxicity (Clifford et al 1991; Ijomah et al 1991).

A survey of scombrotoxic fish poisoning in Britain by Bartholomew et al (1987)
suggests that most cases may be uncomplicated histamine poisoning, and that other
toxins may be involved when the suspect fish contains little histamine, or when
symptoms are not typical. Between 1976 and 1986, Bartholomew et al (1987)
followed up 258 incidents of suspected scombrotoxic fish poisoning. Of 240 fish
samples from these incidents, 101 contained >5 mg histamine/100 g fish. The
symptoms most consistently reported were rash, diarrhoea, flushing and headache. In
any one incident, the symptoms of all patients were similar, although each patient did
not experience all symptoms. Of fish with >20 mg histamine/100 g, 94% were from
incidents in which scombrotoxic symptoms were characteristic. Where fish contained
only 520 mg histamine/100 g fish, only 38% of incidents were 'clinically distinctive'.
The authors regarded rash, flushing and burning of the mouth as distinguishing
features of scombrotoxic fish poisoning. Although gastrointestinal symptoms
especially diarrhoea were frequently experienced, in contrast with some previous
reports (Arnold and Brown 1978), gastrointestinal symptoms alone were not regarded
as indicative of scombrotoxic fish poisoning. Surprisingly, 36/93 fish samples (39%)
with <5 mg/100 g histamine also gave rise to characteristic scombrotoxic (or HFP)
symptoms. Analyses were not done for other biogenic amines, which may have been
toxic in their own right, or histamine potentiators.

Til et al (1997) examined the acute and subacute toxicity of five biogenic amines
tyramine, spermidine, spermine, putrescine and cadaverine in Wistar rats. For the
acute studies, the amines were administered orally as a 25% aqueous solution at
various dose levels to 813/week-old rats (2/sex/group). The rats were observed for
14 days and clinical signs and deaths were recorded. The rats that died and the
survivors killed after 14 days were examined pathologically. For the subacute studies
the rats were fed diets containing 0, 200, 2000 or 10 000 ppm (or mg/kg food)
cadaverine or tyramine; 0, 200, 2000 or 5000 ppm spermine or putrescine; or 0, 20,
200 or 500 ppm spermidine for 56 weeks. The major findings are summarised in the
table below.

15
Summary of major findings obtained in acute and subacute studies
with five biogenic amines

Observations Tyr Spd Spm Put Cad
Approx. LD
50
(mg/kg)
NOEL (ppm)
Mortality
Paralysis
Bodyweight
Anaemia
Haemoconcentration
Impaired renal function
Histopathology
Liver
Kidneys
Heart
>2000
2000
0
0

0
0
0

0
0
0
600
1000
0
0

0
0
0

0
0
0
600
200
++
+

+
0
+ +

+ +
+
+
2000
2000
0
0

0
0
0

0
0
0
5000
2000
0
0

0
+
0

0
0
0
Tyr = tyramine; Spd = spermidine; Spm = spermine; Put = putrescine; Cad = cadaverine
LD
50
= single (oral) dose required to cause death in 50% of animals dosed
NOEL = no observable effect level
0 = not affected; + = slightly increased or slightly affected; = slightly decreased; = moderately
decreased; = markedly decreased.

The doses of biogenic amines tested and tolerated in rats are many times higher than
would be expected in humans following the ingestion of a meal of spoiled fish.
However, the amines were tested individually, not in combination with one another,
or with histamine. Spermine was the most toxic of the five amines tested, causing
liver changes, elevated activity of plasma enzymes associated with the liver,
nephrotoxicity and myocardial degeneration. It is somewhat reassuring that Mietz and
Karmas (1977) found that levels of spermine and spermidine, the most toxic amines in
this series, tended to fall and sometimes reached zero during the decomposition
process in tuna.

2.4.2 Inhibition of histamine detoxification by histamine potentiators

2.4.2.1 Background

Among the scientists who have postulated that histamine is potentiated by some other
component(s) in the toxic fish are Bjeldanes et al (1978), Taylor and Lieber (1979),
Chu and Bjeldanes (1981), Lyons et al (1983), Taylor (1986, 1988), and Stratton et al
(1991). By definition, such a potentiator(s) would act to decrease the threshold dose of
histamine needed to provoke an adverse reaction in humans challenged orally. Certain
drugs have definitely been implicated as contributing factors in cases of histamine
poisoning (Chin et al 1989; Stratton et al 1991).

Taylor (1986) stated that doses of pure histamine required to produce mild reactions
were several times higher than the doses producing more severe symptoms when
consumed with spoiled fish. In the same paper he stated that the variability in
histamine levels in spoiled fish makes estimates of the toxic threshold difficult to
obtain and quoted an estimated threshold dose of histamine in fish at about
60 mg/100 g flesh. Even after allowing for variability in human susceptibility and
variable histamine content in different parts of fish (Lerke et al 1978), it seems that
16
there is indeed some difference between the relative lack of toxicity of pure histamine
and the (often) apparent toxicity of histamine in spoiled fish.

In support of the histamine-potentiator hypothesis, several in vivo and in vitro studies
have suggested that the absorption, metabolism, and/or potency of one biogenic amine
might be modified in the presence of a second amine (Bjeldanes et al 1978; Taylor
and Lieber 1979; Lyons et al 1983). The biogenic amines putrescine and cadaverine
occur in appreciable quantities in toxic fish (Arnold and Brown 1978) and at low
levels in non-toxic fish (Mietz and Karmas 1977). When given in higher ratios
relative to histamine than those that usually seem to occur in toxic fish, these amines
potentiate the biological activity of histamine in laboratory animals.

However, according to Hui and Taylor (1985), uptake of unmetabolised histamine
alone would not be sufficient to elicit some of the symptoms observed in HFP. To
exert its full toxic effects, histamine must reach the peripheral tissues. According to
these authors, the detoxification of histamine in extra-intestinal tissues must also be
inhibited to achieve the full effects.

2.4.2.2 In vitro studies

Mongar (1957) observed that cadaverine and putrescine competitively inhibited DAO
and potentiated histamine-induced contractions in guinea pig ileum. Taylor and Lieber
(1979) tested the effects of 38 chemicals (mainly nitrogen-containing bases) likely to
be consumed with tuna on the in vitro activity of rat jejunal mucosal HMT and DAO.
The most potent inhibitors (at 10 mM concentrations) were monoamines, diamines
and guanidines, including tyramine, -phenylethylamine and tryptamine, but
correlations between chemical structure and inhibitory activity were difficult to
define. The scientists reported that many of the identified chemicals are found in
spoiled tuna, along with histamine, and their ability to inhibit intestinal histamine
catabolism could magnify the oral toxicity of histamine and explain its apparently
greater toxicity when consumed with spoiled tuna.

Cadaverine and aminoguanidine were both strong inhibitors of rat intestinal DAO and
HMT in vitro (cadaverine, 87% and 35%; and aminoguanidine, 100% and 81% for
DAO and HMT respectively). Putrescine was a weak inhibitor (11%) of HMT only
(Taylor and Lieber 1979). Lyons et al (1983) subsequently showed that cadaverine
and aminoguanidine enhanced the absorption of unmetabolised histamine in perfused
rat intestinal segments by inhibiting the conversion of histamine to less-toxic
metabolites. Aminoguanidine is not known to occur in spoiled fish (Lyons et al 1983).

2.4.2.3 In vivo studies

Parrot and Nicot (1966) found that the oral toxicity of histamine in the guinea pig was
increased 10 times when it was administered 40 min after oral administration of
putrescine. And Bjeldanes et al (1978) found that the oral toxicity of histamine in the
guinea pig was potentiated by simultaneous administration of cadaverine. Other
biogenic amines that may act as potentiators of histamine toxicity include tyramine (a
pressor amine that inhibits MAO), tryptamine (which inhibits DAO), and -
17
phenylethylamine (a DAO and HMT inhibitor) (references cited by Stratton et al
1991).

However, Bjeldanes et al (1978), in an article on the aetiology of HFP, put the
question of a synergist for histamine in proper perspective. They pointed out that, to
exhibit synergism, as measured by LD
50
values in the guinea pig, Parrot and Nicot
(1966) used an unrealistically high ratio of putrescine to histamine (5:1, compared
with 1:100 in toxic fish). In their own LD
50
experiments on guinea pigs, Bjeldanes et
al (1978) found that cadaverine was synergistic only at a ratio to histamine of 1:5 or
greater, whereas in toxic fish (tuna) the ratio is 1:10 (Mietz and Karmas 1977; Kim
1978). They also demonstrated that while optimal cadaverine synergism was obtained
by simultaneous administration with histamine to guinea pigs, putrescine required a
time lag before histamine was administered.

As it stands, the above study suggests that it is unlikely that putrescine in spoiled fish
acts as a histamine potentiator, and that cadaverine may only possibly be involved.
However, Klausen and Lund (1986) found, when comparing mackerel and herring,
that levels of cadaverine in mackerel may exceed those of histamine by 25 times,
which may well explain why this species commonly causes HFP.

Hui and Taylor (1985) determined the effects of enzyme inhibitors of histamine in
vivo and their possible role in potentiating histamine toxicity by studying the effects of
known inhibitors of DAO, HMT and MAO, both foodborne and pharmacological, on
the urinary excretion pattern of histamine and its metabolites in rats. When they
administered [
14
C]-histamine orally to rats (female Sprague-Dawley, 35/group), an
average of 80% of the administered radioactivity was recovered in the urine after 24 h.
About 10% of the total dose was excreted via the faeces. Analysis of 4-h urine
samples showed imidazoleacetic acid was the predominant metabolite (60.6%), and
N-methyl-imidazoleacetic acid (8.6%), N-methylhistamine (7.3%) and N-
acetylhistamine (4.5%) were minor metabolites.

Histamine metabolism was inhibited by simultaneous oral administration of the drugs
aminoguanidine, isoniazid and quinacrine, and the biogenic amines cadaverine,
putrescine, tyramine and -phenylethylamine. The administration of inhibitors
resulted in an increased amount of unmetabolised histamine and a decreased amount
of metabolites reaching the urine. However, the total rate of excretion of histamine
and its metabolites in the urine was similar in the presence and absence of
potentiators, indicating that the total amount of absorption and excretion of histamine
and its metabolites was equivalent, with or without potentiators.

Hui and Taylor (1985) found that pharmacological inhibitors were more potent and
had a longer duration of action in rats than foodborne ones. Cadaverine
dihydrochloride (2 mmol/kg, equivalent to 204 mg cadaverine/kg), putrescine
dihydrochloride (2.5 mmol/kg), tyramine hydrochloride (2.5 mmol/kg) and -
phenylethylamine hydrochloride (2.5 mmol/kg) were weak inhibitors, effective at
doses 45 times higher than that of simultaneously administered histamine
dihydrochloride (0.5 mmol/kg, equivalent to 55 mg histamine/kg).

18
Inhibitors of DAO (aminoguanidine, isoniazid, cadaverine, -phenylethylamine and
tyramine) had a more profound effect on excretion of unmetabolised histamine in rats
than inhibitors of only HMT. To test the possibility that more than one potentiator
may be involved in foods implicated in histamine poisoning incidents, Hui and Taylor
(1985) tested a mixture of histamine (0.5 mmol/kg) and the foodborne inhibitors
cadaverine, putrescine, tyramine, -phenylethylamine and tryptamine (each dosed at
0.5 mmol/kg). The mixture increased the amount of unmetabolised histamine in 4-h
urine samples 100% over that present when histamine was dosed alone.

These results indicate that cadaverine and these other amines could indeed potentiate
histamine toxicity in spoiled fish if present alone (in the case of cadaverine) or
collectively at levels 4 times greater than that of histamine. Furthermore, it is likely
that all the possible histamine potentiators have not been identified.

2.4.3 Barrier disruption hypothesis

Another possible mechanism of potentiation of histamine by other biogenic amines is
the barrier disruption hypothesis. The hypothesis was first proposed by Parrot and
Nicot (1966), who suggested that potentiators might interfere with the protective
actions of intestinal mucin. Intestinal mucin is known to bind histamine, and it has
been suggested that this binding is essential to prevent the intestinal absorption of
histamine. Potentiation would occur by disruption of the bonding and resulting
enhancement of absorption and an increase in histamine toxicity (Jung and Bjeldanes
1979; Chu and Bjeldanes 1981).

Jung and Bjeldanes (1979) found that cadaverine exhibited a marked influence on the
rate of transport of
14
C-labelled histamine and metabolites across the gut wall in
isolated gut sections of the guinea pig. However, cadaverine had a minor effect on
histamine metabolism: the percentage of free histamine appearing outside the gut sac
increased from 30.7% when histamine alone was present, to 35.2% and 37.2% when
cadaverine was added in ratios to histamine of 1:2 and 1:1 respectively. When
histamine alone was placed inside the gut, 41.2% of radioactivity was transported
across the gut wall as histamine and metabolites. The percentage increased
significantly to 48% by adding cadaverine in the ratio of cadaverine:histamine of 1:10,
to 52.8% at the ratio 1:2, and to 53.6% at the ratio 1:1.

Jung and Bjeldanes (1979) suggested that cadaverine potentiation of histamine
toxicity may result from induction of increased rates of absorption of histamine and
metabolites, and that the established antihistaminase activity of cadaverine does not
appear to play a significant role. They believed their results were consistent with a
proposed role of substances such as mucin in maintaining a barrier to histamine
transport.

According to Taylor (1986) and Mitchell (1993), experiments based on this theory
have not so far provided a convincing rationale for the apparent toxicity of histamine
in fish.

19
2.4.4 Release of endogenous (mast cell) histamine by scombroid toxin(s)

In 1957, Mongar had reported that diamines in the series NH
2
(CH
2
)
n
NH
2
release
histamines from isolated tissues. His results were obtained using minced guinea pig
lung and isolated rat diaphragm to measure histamine-releasing activity of diamines of
chain length C
5
C
15
. The activity increased steadily with chain length from less than
5% release with cadaverine to >90% release with pentadecamethylene-diamine during
a test period of 15 min. The effect of all diamines increased steeply with
concentration. In a concentration of 1 mM, cadaverine was inactive both on minced
guinea-pig lung and rat diaphragm, but, at 100 mM, it released about 80% of the
histamine content of the tissue.

As mentioned above, Mongar (1957) also reported on the antihistamine activity of
diamines, which produced a depression of the histamine contractions of guinea-pig
ileum. Histamine-releasing activity increased steadily with chain length, but the
activity of diamines as inhibitors of histaminase decreased with chain length. Mongar
(1957) noted that the parallelism between histamine-releasing activity and toxicity
extended to monoamines in the series CH
3
(CH
2
)
n-1
NH
2
. Histamine-releasing activity
of both series increased with pH and was attributed mainly to the non-ionised base.
However, since the compounds were inactive at pH 7 and their activity rose sharply
with pH to reach a high value at pH 8.5, they would be ionised at physiological pH
and not active in producing endogenous histamine in the human body.

However, the idea that release of endogenous histamine may be involved in HFP
persisted. Attention began to focus on mast cells, the granules of which contain a
histamineheparin complex (Riley 1959). In 1972, Olley postulated that 'some other
basic substance produced during spoilage [of fish] may release histamine from the
histamineheparin complex of mast cells'. Arnold and Brown (1978) also pointed out
that release of histamine from mast cell granules could account for some, if not all, of
the symptoms associated with scombroid toxicity.

Subsequently, Clifford et al (1991) and Ijomah et al (1991) of the UK Ministry of
Agriculture, Fisheries and Food postulated that the scombroid toxin(s) is a mast cell
degranulator, and the antihistamine therapy is effective because it eliminates the effect
of endogenous histamine.

Ijomah et al (1991, 1992) fed mackerel deliberately exposed to poor storage
conditions to volunteers. They found that the volunteers fell into susceptible and non-
susceptible subgroups, and that the level of histamine in fish did not correlate with
toxicity exhibited. However, when subjects were dosed with either placebo or H
1
antagonist (chlorpheniramine 4 mg), the antihistamine convincingly abolished
vomiting and diarrhoea associated with the ingestion of scombrotoxic fish. The
scientists concluded that dietary histamine played a minor role in the toxicity and that
the major contributor was a postulated agent that released histamine by degranulation
of mast cells in the gastrointestinal tract.

Clifford et al (1991) went further, to analyse mackerel fillets that had been associated
with an outbreak of 'scombrotoxicosis' for their contents of cadaverine, histamine,
putrescine, spermidine, spermine and tyramine. The same fillets were fed to healthy
20
volunteers. Susceptibility to the disease was quite variable. Of the 86 fillets examined,
30 rapidly induced nausea/vomiting and/or diarrhoea when 50-g portions were
consumed. The remaining fillets failed to provoke such symptoms, even though
volunteers proven to be susceptible to scombrotoxicosis tested 17 of them. Statistical
analysis failed to detect differences in amine content between fillets shown to be
scombrotoxic and those failing to induce symptoms, and failed to establish significant
relationships between the amine doses and volunteer responses, even after
manipulations to simulate additive or synergistic interactions. The minimum doses
associated with scombrotoxicosis and the maximum doses giving negative responses
in susceptible volunteers were: histamine, 0.23 and 3.33; cadaverine, <0.02 and 0.38;
putrescine, 0.0008 and 0.01; tyramine, 0.009 and 0.75 mg/kg bodyweight (bw)
respectively. The scientists concluded that the contents of such amines in mackerel
have little or no role in the aetiology of scombrotoxicosis, even when acting additively
or synergistically, and that the primary scombrotoxin, which remained unknown, was
responsible for mast cell degranulation.

Other clinical reports of HFP, for examples those caused by the consumption of
salmon, support the suggestion that exogenous histamine is not always responsible for
intoxication, and that other toxins and/or endogenous histamine may be involved.
Bartholomew et al (1987) reported six incidents of scombrotoxic fish poisoning in
canned salmon in Britain in 1983. In five of the incidents, the incriminated fish
contained <1 mg/100 g histamine, despite the presence of characteristic symptoms and
incubation period of histamine poisoning. Salmon from the sixth episode contained
only 17 mg/100 g histamine.

In addition, Gessner et al (1996) described a typical and severe scombrotoxic-like
illness following the ingestion of smoked sockeye salmon that demonstrated low
histamine levels and high toxicity on mouse bioassay. Symptoms included flushing,
pruritus, nausea, sweating, vomiting, diarrhoea, dizziness etc. The implicated salmon
had histamine levels 25-fold less than the US Food and Drug Administration (FDA)
toxicity level for tuna of 50 mg/100 g (FDA 1998). The patient ate an estimated
0.0006 mg of histamine/kg bw far less than the estimated 1 mg of histamine/kg bw
reported to cause human illness (Taylor 1986). Estimated levels for putrescine and
cadaverine were 0.67 mg/100 g and 0.19 mg/100 g. (The analyses were performed on
fish caught and treated at the same time and in the same way as the incriminated fish.)

Some imidazole compounds are known to release histamine from mast cells by a non-
immunological mechanism; for example, imidazole fungicides (Gietzen et al 1996). It
is interesting to note that urocanic acid, an imidazole compound and a histidine
metabolite of spoiling fish (Kawai and Sakaguchi 1968; Mackie and Fernndez-
Salguero 1977; Baranowski 1985), has recently been described as an inducer of
histamine in vivo in mice (Hart et al 1997) and as a mast cell degranulator in human
skin organ cultures (Wille and Kydonieus 1995; Wille et al 1999). Urocanic acid may
be one of the 'toxic imidazole compounds' in spoiling fish mentioned by Olley in 1972
and the 'scombroid toxin' that researchers have been seeking for decades.

Some experimental evidence seems to reject the mast cell degranulation theory, but
this may reflect the large number of variables involved in the pathogenesis of HFP.
Mast cell degranulation may not occur in all cases of the disease, but may be an
21
important feature in other cases. When Van Gelderen et al (1992) gave eight healthy
volunteers either herring paste containing 90 mg histamine formed from induced
spoilage with photobacteria or fresh fish to which 90 mg histamine had been added,
the maximum histamine concentration in plasma did not differ significantly between
the two dose regimes. However, herring may not have been the most suitable fish to
use in this experiment. Klausen and Lund (1986) found that herring contained 45
times less free histidine and free lysine (cadaverine precursor) than fresh mackerel. In
addition, the photobacteria used to induce spoilage may not have possessed HAL,
which is required to produce urocanic acid from histidine.

Morrow et al (1991) measured the urinary excretion of histamine and its metabolite,
N-methylhistamine, in three people who had HFP after eating marlin that contained
high levels of histamine (8422503 mol/100 g, equivalent to 93278 mg/100 g).
They also measured 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-
dioic acid (PGD-M), the principal metabolite of prostaglandin D
2
, a mast cell
secretory product, to assess whether mast cells had been activated to release
histamine. Flushing and headache began in all three people 1030 min after ingestion
of the fish. One person also had severe diarrhoea of sudden onset. The levels of
histamine and N-methylhistamine were 920 and 1520 times respectively the normal
means in urine samples collected 14 h after the meal. During the subsequent 24 h,
the levels fell to 415 and 411 times the normal values, and 14 days later they had
returned to normal. PGD-M excretion was not increased at any time. Two of the three
patients were treated with an antihistamine (diphenhydramine), which was rapidly
effective.

In the study of Morrow et al (1991), HFP was associated with urinary excretion of
histamine in quantities far exceeding those required to produce toxicity. The high
histamine levels in the incriminated fish, together with the failure to find increased
endogenous release of prostaglandin D
2
, indicated that the histamine was derived from
the fish. Analyses were not done for other biogenic amines, which could have been
histamine potentiators. However, increases in the urinary excretion of both histamine
and N-methylhistamine of similar magnitude in the poisoned people suggested that the
spoiled fish did not contain a substance or substances that potentiated histamine
toxicity by inhibiting its inactivation by HMT.

Like Morrow et al (1991), Snchez-Guerrero et al (1997) rejected the proposal that
endogenous histamine is involved in HFP. The latter authors used immunoassay to
measure tryptase, another indicator of mast cell degranulation, in seven patients
intoxicated after eating tuna. The quantification of tryptase release in vivo allows a
precise valuation of mast cell activation, as the enzyme is located in secretory granules
of mast cells and released at the same time as histamine during degranulation. The
low levels obtained caused the authors to reject an anaphylactic-type reaction and
favour poisoning by an exogenous source of histamine (fish).

The role of endogenous histamine release, if any, in histamine/scombroid fish
poisoning remains uncertain and unproved. The poisoning syndrome described by
Ijomah et al (1991, 1992) and Clifford et al (1991) was not what would be considered
typical of HFP. Many toxins produce nausea, vomiting and diarrhoea. Mitchell (1993)
wrote that a possible factor in explaining the apparent contradiction of this work with
22
previous observations on HFP is the definition of poisoning that was applied. Ijomah
et al (1991) considered that vomiting and/or diarrhoea were essential factors in
poisoning. Flushing and tingling in the mouth were considered minor and possibly
caused by dietary histamine. The syndrome described by Ijomah, Clifford and
colleagues may well have been complicated by a toxin or toxins additional to those
commonly associated with HFP. The complexity of fish poisoning makes it difficult
to do clinical trials on HFP. Unless there is consensus of opinion on the clinical signs
and symptoms of HFP, such trials are of little value.

2.4.5 Are paralytic and diarrhetic shellfish poisons involved?

Clifford et al (1993) found low levels of paralytic shellfish poisons (saxitoxins) in
toxic mackerel (0.021.30 g saxitoxin equivalents/kg) by enzyme-linked
immunosorbent assay (ELISA) and suggested that, possibly in combination with
diarrhetic shellfish poisons, they may be responsible for scombroid fish poisoning.
These concentrations of saxitoxin equivalents corresponded to doses in the range
0.041.0 ng/kg bw and were orders of magnitude less than the intraperitoneal and oral
LD
50
values in mice (10 and 263 g/kg respectively).

Calculations made following an outbreak of mussel poisoning in England suggested
that doses as low as 9 g saxitoxin equivalents/kg bw may cause illness in humans,
but doses as high as 86 g/kg bw are not necessarily fatal (references cited by Clifford
et al 1993). Nine of 23 mackerel samples associated with scombrotoxic incidents
apparently contained less saxitoxin than the negative control sample, and dose
(saxitoxin equivalents) could not be correlated with the volunteer response. Doses
associated with nausea/vomiting and/or diarrhoea ranged from 0.11 to 1.0 ng/kg bw,
whereas doses not producing these symptoms ranged up to 0.5 ng/kg bw. Clifford et
al (1993) concluded that paralytic shellfish poisons are at least partly responsible for
'scombrotoxicosis'. However, the observation during volunteer testing of nausea and
vomiting at doses orders of magnitude less than previously reported for saxitoxin,
coupled with the presence of diarrhoea, which is not characteristic of paralytic
shellfish poisoning, suggest that this is unlikely.

It would appear that a clear definition of histamine/scombroid fish poisoning is
required, based on the clinical signs/symptoms described by Taylor (1985) and Wu et
al (1997) below. Many instances of fish poisoning are probably the result of a number
of unrelated toxins acting in concert. As such, they are not typical of
histamine/scombroid fish poisoning, and should not be given that classification. Much
research is needed to sort out the many toxins in spoiling fish and the syndromes they
cause.

2.4.6 Absorption of histamine from mouth and throat

An unusual hypothesis (cited by Lange 1988) is that when affected fish are eaten the
histamine may be absorbed through the mucous membranes of the mouth and throat,
thus bypassing the digestive process that destroys it. It is difficult to propose a
mechanism whereby a fish substrate would enhance the absorption of histamine
through mucous membranes.

23
2.5 Clinical characteristics and treatment

2.5.1 Clinical signs and symptoms

Signs and symptoms of HFP occur from several minutes to several hours after
ingestion of the toxic fish. The illness typically lasts a few hours, but may continue for
several days. The primary signs/symptoms are those of histamine poisoning
cutaneous (rash, urticaria, oedema and localised inflammation), gastrointestinal
(nausea, vomiting, diarrhoea), haemodynamic (hypotension) and neurological
(headache, palpitations, tingling, burning, itching) (Taylor 1985; Wu et al 1997). In
severe cases there may be bronchospasm and respiratory distress (Shalaby 1996).
The most consistent clinical sign reported by Arnold and Brown (1978) was flushing
of the skin of the face and neck, which caused a feeling of intense heat and general
discomfort. Because this striking erythema predominantly involves exposed areas, it
resembles sunburn (Kim 1979). Gastrointestinal symptoms were experienced by fewer
than 25% of victims described by Arnold and Brown (1978). When present, they
generally took the form of nausea and diarrhoea. However, when Gilbert et al (1980)
reported on 150 patients affected in 30 separate outbreaks of 'scombrotoxic fish
poisoning' in Britain, in each outbreak the symptoms were similar, and diarrhoea was
predominant. Symptoms were recorded for each incident rather than individually, and
a breakdown of patient numbers was not provided. The following table lists the
number of outbreaks in which particular symptoms were noted.

Signs and symptoms associated with scombrotoxic
fish poisoning in 30 outbreaks (Gilbert et al 1980)

Sign or symptom No. of outbreaks
Diarrhoea
Hot flush/sweating
Erythematous rash
Nausea
Headache
Abdominal pain
Palpitations
Burning in mouth
Vomiting
Feverish
Giddy or dizzy
Tight chest
Respiratory distress
Facial swelling
24
15
14
13
13
6
6
5
4
3
2
1
1
1

Diarrhoea was also a prominent clinical sign in 77% of patients (second in frequency
only to skin rash, which occurred in 82%) in ten incidents of HFP involving 22
patients in South Africa (Mller et al 1992). Cape yellowtail (Seriola lalandii), a non-
scombroid fish, was involved in all cases. Other signs were palpitations, headache,
nausea and abdominal cramps, paraesthesia, an unusual taste sensation and breathing
difficulties. The patients responded well to antihistamine therapy and most had
recovered within 1224 h.

24
Wu et al (1997) described two outbreaks of HFP that occurred in Taiwan in 1996 and
were confirmed by toxin analysis. In the first, caused by a non-scombroid fish, the
main symptoms were facial flush, dizziness, headache, conjunctival hyperaemia and
hypotension. In the second outbreak, in which a scombroid fish was involved,
dizziness, diarrhoea, flushing and headache were the most common symptoms. Levels
of histamine and cadaverine in fish samples taken from the two outbreaks were 84 and
8.5 mg/100 g (1st outbreak) and 272 and 23 mg/100 g (2nd outbreak) respectively.

Anxiety is often mentioned as a prominent symptom of HlP (e.g. Russell and Maretic
1986; Specht 1998; Sabroe and Kobza Black 1998). A case of transient loss of vision
in association with atrial tachycardia with a 2:1 atrioventricular block was also
reported. Other signs and symptoms in this patient were typical of HFP, and response
to antihistamine therapy, including return of vision, was rapid (McInerney et al 1996;
Clark 1997).

In 1997, Ascione et al reported two cases of scombroid syndrome in Palermo, Italy,
following ingestion of cooked tuna. Apart from symptoms and clinical signs typical of
HFP, both patients exhibited cardiovascular shock. This was associated with
subendocardial myocardial infarction in one case and acute pulmonary oedema with
myocardial ischaemia in the other.

There appears to be no information on the effect of HFP in a woman on an
embryo/foetus or breastfed infant.

2.5.2 Diagnosis

A diagnosis of HFP depends on a history of fish eaten by the patient immediately
before onset of the illness. If the symptoms are typical of histamine poisoning, the
onset time is short, and the patient has eaten a type of fish that has previously been
implicated in cases of histamine poisoning, then a tentative diagnosis of HFP can be
made. The diagnosis can be confirmed by detecting high levels of histamine in the
contaminated food, meal remnants or in a similar fish obtained from the same source
(Taylor 1986).

Apart from being somewhat inconsistent, the symptoms of HFP are not definitive.
Many can occur with other illnesses, both foodborne and non-foodborne. When
diarrhoea is the predominant symptom, histamine may not be the main toxin involved.
In addition, histamine poisoning is often confused diagnostically with food allergies.
Identical symptoms occur, and antihistamines are equally effective in treating both
illnesses. However, histamine poisoning can easily be distinguished from food allergy
on the basis of: the lack of a previous history of allergic reactions to the incriminated
food; the high attack rate in group outbreaks; and the detection of high levels of
histamine in contaminated food. With the fish that are commonly associated with
HFP, histamine poisoning would be encountered much more frequently than allergic
reactions. IgE-mediated allergic reactions could be detected by using skin prick tests
with extracts of similar fish with known low histamine content (Taylor 1986).

25
2.5.3 Treatment

Symptoms of HFP rapidly subside after antihistamine treatment. H
1
antagonists, such
as diphenhydramine or chlorpheniramine, are usually selected, but H
2
antagonists such
as cimetidine may also be effective (Guss 1998). Induced emesis has also been
recommended as a treatment (Downs 1997). Since the disease is self-limited, running
a short course, pharmacological intervention is not always necessary (Taylor 1986).

2.5.4 Clinical complications

Serious complications are rarely encountered in cases of HFP. However, on rare
occasions, serious cardiac and respiratory complications occur in individuals with pre-
existing cardiac and respiratory conditions (Taylor et al 1989). For example, Russell
and Maretic (1986) described the case of a young child vith a history of bronchial
asthma, who suffered respiratory collapse after consuming fish contaminated with
excessive amounts of histamine.

To determine whether kidney disease could exacerbate the symptoms of HFP,
Helander et al (1965) investigated the renal removal of histamine in a person with
severely impaired renal function caused by chronic pyelonephritis. The percent renal
extraction of injected [
14
C]-histamine from whole blood was reduced (to 51%)
compared with the values of 7486% obtained in healthy subjects.

2.6 Detection of histamine and other biogenic amines in fish

2.6.1 Detection of histamine-producing bacteria

Many methods have been developed to detect histamine-producing bacteria in foods
such as fish. Most of these detection systems are based on specific media that are
selective and/or differential for histamine-producing bacteria (e.g. Niven et al 1981).
Some methods require the preliminary isolation of bacteria so that individual isolates
can be assessed for histamine formation. One system utilises a histidine-fortified
trypticase soy broth that is optimised for bacterial histamine production (Taylor and
Woychik 1982). Another utilises a tuna fish infusion broth (Omura et al 1978), but
this is a poorly defined medium and dependent on the availability of high quality raw
tuna (Taylor and Woychik 1982). In these procedures, the histamine is extracted and
measured by fluorometric assay.

Another system for the detection of histamine-forming bacteria in fish is a differential
medium containing bromocresol purple. Histamine formation by a colony causes a
colour change due to a shift in pH. MRS broth fortified with histidine can be used in
the identification of histamine-producing lactobacilli. The histamine can be extracted
and measured fluorometrically, or a procedure based on diamine oxidase and
leucocrystal violet can be applied directly to the growth medium (references cited by
Stratton and Taylor 1991).

However, there have been problems with identification of histamine-producing
bacteria. Taylor et al (1978a) listed bacteria previously identified as histamine
producers, but found that evidence firmly linking some of the bacterial species on that
26
list to histamine production was lacking. They considered that discrepancies could be
due to differences between strains within a particular species, growth media, or
analytical methodology used in the detection of histamine or HD. For example,
Nivens medium (Niven et al 1981) has been associated with false positive results.
Garland (1985) attributed this to the non-specific alkaline products generated through
the metabolism of tryptone and yeast extract by bacteria. McMeekin (1986) suggested
that the incorporation of glucose at 0.1% in the medium could balance non-specific
alkaline products. However, this medium still gave problems with M. morganii that
suggested that the amount of acid formed from glucose produced by different
organisms was different, depending on the bacterial glucose fermentation pathway.
When 0.1% maltose was incorporated by Anggawati (1986), most false positive
results were eliminated (Leung 1987).

According to Taylor (1986), many early studies of bacterial histamine production were
not comparative, making it difficult to determine whether histamine production by
bacteria was prolific or inconsequential. A reason for measuring histamine production
by bacteria rather than HD activity is that several bacterial species also produce
histaminase, which may limit histamine accumulation by these species in foods
(Ienistea 1971).

Klausen and Huss (1987a) developed a method based on automated conductance
measurements in a histidine-containing medium incubated at 25
o
C. The method is
rapid, with histamine-producing bacteria being detected within 24 h. Such a method
could be used to screen large numbers of fish samples that may be contaminated with
significant amounts of histamine-producing bacteria, and thus could be part of a
quality assurance program (Klausen and Huss 1987a). However, the method has the
disadvantage of requiring expensive instrumentation (Stratton and Taylor 1991).

An improved test for screening for histamine-producing bacteria is required, ideally
encompassing and distinguishing cadaverine- and putrescine-producing bacteria
(James and Olley 1985).

2.6.2 Analysis of histamine

Researchers in the field, as well as industry representatives, unanimously agree that
canned tuna with high levels of histamine imparts a peppery feel to the mouth when
chewed, but tasting on a routine basis is not a feasible means of quality assurance
(Etkind et al 1987). There is considerable evidence associating several other
measurements of fish deterioration with histamine levels. For example, Cantoni et al
(1976) compared histamine production with that of volatile acids and volatile bases in
tuna stored at 1820
o
C. Toxic levels of histamine (>100 mg histamine/100 g fish)
were noted after 4 days. During the same 4-day period, high levels of volatile acids
and bases were also noted. Hungerford et al (1997) developed a simple field test for
decomposition of seafood based on volatile bases. However, no objective
measurement, short of determining histamine concentration, has emerged as an
effective indicator of histamine levels.

The classical method for determination of histamine was based on the fact that it will
cause contraction of guinea pig ileum (Arnold and Brown 1978). The first application
27
of this method to the analysis of histamine in fish was reported by Geiger (1944a). He
and fellow workers had earlier identified a biologically active substance in marine fish
as histamine (Geiger et al 1944). Geiger (1944a) detailed how the bioassay could be
applied to fish and reported findings on histamine levels in raw and canned sardines
and mackerel. He pointed out that canning did not interfere with subsequent analysis
for histamine, and demonstrated the practical application of histamine determination
in canned tuna.

Chemical testing is an effective means of detecting the presence in fish muscle.
However, the validity of such testing depends on the design of the sampling plan. For
this reason, chemical testing alone will not normally provide assurance that the hazard
has been controlled. Because histamine is generally not uniformly distributed in a
decomposed fish, a guidance level of 50 ppm has been set (FDA 1998). If 50 ppm is
found in one section, there is the possibility that other sections may exceed the
toxicity level of 500 ppm (FDA 1998). Additionally, recent studies suggest that, if HD
levels are high, histamine formation can continue even in frozen storage (Price 1999).

Two other problems remain. One is the lack of standardisation of histamine detection
methodology, with numerous different methods being used around the world; and the
second is that the presence of histamine alone is not necessarily a reliable indicator of
fish likely to cause HFP.

Since the guinea pig ileum method of Geiger (1944a) for histamine in unprocessed
and canned fish, many diverse analytical procedures have been published. Some of the
more important include tests for histamine in:

fish products: thin layer chromatographic method (Shultz et al 1976)
canned tuna: fluorometric method (Lerke and Bell 1976)
fish: fluorometric method (Taylor et al 1978b)
fish: enzyme-based screening test (Lerke et al 1983)
seafood: flow-injection method (Hungerford et al 1990)
canned fish: HPLC (Yen and Hsieh 1991)
fish: capillary electrophoresis (Mopper and Sciacchitano 1994)
tuna: copper chelation method (Bateman et al 1994)
fish: oxygen-sensor-based method (Ohashi et al 1994)
seafood: biological method (Association of Official Analytical Chemists, AOAC,
1995a)
seafood: chemical method (AOAC 1995b)
seafood: fluorometric method (AOAC 1995c)
seafood: modification of AOAC method (Rogers and Staruszkiewicz 1997).

Historically, determination of histamine has been difficult due to the lack of rapid
testing methods. However, rapid testing methods are now available (Hungerford et al
1997; Miller et al 1997; Price 1999). For example, commercial competitive ELISA
kits are available for histamine detection, such as ALERT
TM
(sensitivity 550 ppm,
qualitative, 2 h) and Veratox
TM
(sensitivity <5 ppm, quantitative from 050 ppm, 1 h)
from Neogen Corporation, USA (and Elisa Systems, Australia). There are also several
enzyme immunoassay test kits, including Histamarine Test Kit (sensitivity 0.5 ppm,
28
quantitative from 1500 ppm, 1 h) from Immunotech, France, approved by the
Association of Official Analytical Chemists (AOAC) (Price 1999).

In the ELISA kits, histamine is extracted from a sample in a quick water-extraction
process. The extract is filtered, then diluted into a buffer solution prior to running on
the ELISA. These kits provide controls for rapid distinct visual colour results and
eliminate the need for complicated chemical extraction of samples. They provide good
reproducibility and 'low' false negative and false positive rates when tested against the
official AOAC fluorometric method for histamine (Anon. 1999; Rob Sherlock, Elisa
Systems, pers. comm. 1999). However, at more than $10/test, they are still too
expensive for routine testing of numerous samples on a quality-assurance or
preventive-testing basis. They are, however, suitable for disease outbreak
investigatory studies.

2.6.3 Analysis of other biogenic amines and chemical quality index

The assessment of the biogenic amine content in foods has received much attention
for many years due to the possibility of using amine concentrations as an index of
food quality, and has included various HPLC methods besides those for histamine
(Mietz and Karmas 1977; Edwards et al 1987).

Because histamine alone is not always useful as an indicator of fish quality, Mietz and
Karmas (1977) established a chemical quality index of canned tuna for estimating the
extent of decomposition in fresh tuna prior to canning. They used qualitative and
quantitative HPLC to examine the relationship of dansyl derivatives of five amines
(histamine, putrescine, cadaverine, spermine and spermidine) extracted from ground
canned fish. They prepared standards of pure putrescine, cadaverine, spermine and
spermidine at concentrations of 1 mg/100 mL, and the level of each of the five
compounds in canned fish samples was calculated on a ppm basis to generate an index
of tuna decomposition.

Mietz and Karmas (1977) prepared and canned 48 cans of tuna ('authentic pack') as
good, borderline and decomposed; received 6 cans from the US FDA as authentic
pack 'good', 'borderline' and 'decomposed'; and purchased 87 cans of commercial
canned tuna. The tuna included skipjack, yellowfin and albacore. All 141 cans were
evaluated organoleptically after opening and scored from 1 to 10, with the highest
values representing the most decomposition. Subsequently the scores were translated
to good, borderline, and decomposed. The classification of borderline indicated initial
stages of decomposition.

Significant differences were seen between the good, borderline and decomposed tuna.
Low levels of putrescine, cadaverine and histamine were typical of good tuna.
Relatively high levels of spermine and spermidine were likewise found in good tuna.
The borderline tuna showed a significant rise in the levels of putrescine, cadaverine
and histamine, and generally decreased levels of spermine and spermidine. In
decomposed tuna, the putrescine, cadaverine and especially histamine levels
continued to increase significantly, while the spermine and spermidine levels fell, with
many samples showing the absence of these two amines (thus necessitating a factor of
1 in the denominator of the index below). Average values for histamine, putrescine,
29
cadaverine, spermidine and spermine in authentic pack decomposed tuna were 253,
2.5, 19, 0.7 and 1.9 ppm (or g/g) respectively.

Each of the five compounds per fish sample in ppm was submitted to the formula:

Index = ppm histamine + ppm putrescine + ppm cadaverine
1 + ppm spermine + ppm spermidine

Nominal cut-off values were established for the index scores to establish Class 1,
Class 2 or Class 3 fish. The decomposition index followed an exponential value. The
values were Class 1, 01; Class 2, 110; and Class 3, >10. These nominal cut-off
values were found to be effective in discriminating between the classes of fish, and
appeared not to be dependent on species of fish or packing media.

The resulting chemical index scores (see table below) compared favourably to
'authentic pack' and organoleptic value scores.

Organoleptic scores versus index scores for all samples

Index scores (141) Organoleptic scores
(141) Class 1 (109) Class 2 (15) Class 3 (17)
Good (85)
Borderline (36)
Decomposed (20)
73
a
25
11
b
11
4
a
0
1
b
7
9
a
% agreement (a) = 61%
% disagreement (b) = 8.5%
(Borderline samples classified either good or decomposed by either score were not included in the %
agreement or disagreement.)

Since it measures several different compounds resulting from several different
processes of decomposition, this method could be used as a chemical indicator for
decomposition of tuna. However, it is too complex to be used for routine screening.

Wagener (1984) prepared dansyl derivatives of extracted amines from fresh and
spoiled fish using the method of Hui and Taylor (1983) and examined them by HPLC.
He confirmed the earlier findings, that levels of histamine, putrescine and cadaverine
rise rapidly during the initial stages of fish spoilage. He also identified tryptamine in
tuna, hake and mackerel and tyramine in mackerel, but found that levels of these did
not change much during storage. Interestingly, there were unidentified peaks on the
chromatograms, some increasing and some decreasing during spoilage. An unknown
peak marked 'X' showed an increase in both tuna and hake, and was very prominent in
canned mackerel. Wagener (1984) speculated that the peak could be the substance
'saurine' noted by Japanese workers in spoiling saury (Kawabata et al 1955).
(However, Foo identified saurine as the phosphate salt of histamine in 1976.) The
unidentified compound was apparently not tested by Pauly's reagent, and may be the
same as Shewan's compound 8 (Shewan 1955) or Hughes' compound X (Hughes
1959). It is interesting to note that Mietz and Karmas (1977) also found a dansylated
peak (shoulder) between cadaverine and histamine in decomposed canned tuna, which
remained unidentified.

30
In 1997, the US FDA published details of a gas chromatographic (GC) method for
putrescine and cadaverine in seafood. The extracted diamines are converted to
fluorinated derivatives, which are passed through solid-phase extraction columns and
the derivatives quantified by electron-capture GC after separation. Fourteen
laboratories analysed 14 samples of canned tuna and raw mahi-mahi (including blind
duplicates and a spike) containing 0.22.6 ppm putrescine and 0.69.1 ppm
cadaverine using the method. Recoveries ranged from 71 to 102% for putrescine and
from 77 to 112% for cadaverine. AOAC International has adopted the GC method for
determination of putrescine in canned tuna and cadaverine in canned tuna and mahi-
mahi (Rogers and Staruszkiewicz 1997).

More recently, an Italian group has developed an improved method for the
simultaneous determination of underivatised biogenic amines, cadaverine, putrescine,
spermidine, histamine, tyramine and some amino acid precursors, histidine and
tyrosine, in food products. The method is based on ion-exchange chromatography (IC)
with integrated pulsed amperometric detection (IPAD). The method was used
successfully for the analysis of biogenic amines and amino acids in food both of
vegetable and animal origin (pilchards) and in fermented foods, such as cheese and
salami. The main advantages of this method over a previous HPLC method coupled
with IPAD (Draisci et al 1993) are its application to a larger number of analytes and
matrices, a simpler extraction and clean-up procedure, and an improved
chromatographic separation and a lower detection limit. The IC-IPAD method is
suitable for the detection of biogenic amines in a large number of samples and is
particularly useful for routine checks in repetitive analyses (Draisci et al 1998).

2.6.4 Analysis of urocanic acid

Although he did not postulate that urocanic may be a candidate scombroid toxin,
Baranowski (1985) suggested that urocanic acid may be a useful alternative to
histamine as a spoilage index in scombroid and other fish rich in endogenous
histidine. He used thin-layer chromatography to analyse for urocanic acid using
standards in 95% alcohol. Morrison et al (1980) and Caron et al (1982) have used
HPLC methods.

31
3. DOSERESPONSE ASSESSMENT

3.1 Definition of doseresponse assessment

Kindred (1996) defined doseresponse assessment as The determination of the
relationship between the magnitude of exposure and the probability of occurrence of
the health effects in question. Doseresponse assessment is often termed hazard
characterisation (Codex Alimentarius Commission 1996).

The kind of topics that may be discussed under doseresponse assessment include:

the quantity of hazard in relation to the frequency and magnitude of adverse
effects;
susceptibility of the general population and sub-populations;
food composition characteristics that affect hosthazard interaction;
special characteristics of the agent that affect hostpathogen interaction;
rates of intoxication;
morbidity and mortality rates (severity of the disease, sequelae);
human volunteer feeding studies;
epidemiological data;
animal data; and
clinical and laboratory studies.

The data required in this area of risk assessment are largely quantitative rather than
qualitative, to complement the descriptive elements of HFP discussed under Hazard
Identification. Because of the highly variable nature of the epidemiology and clinical
features of the disease, this section is difficult to address. It has been approached by
looking at the incidence of HFP; fish characteristics that affect the clinical response;
amount of bacterial contamination in relation to histamine; bacterial species involved;
concentration of histamine and other biogenic amines in fish and the toxic dose;
human factors that affect the clinical response; and morbidity and mortality rates.

3.2 Incidence of histamine fish poisoning

3.2.1 Background

HFP occurs throughout the world and is perhaps the most common form of toxicity
caused by the ingestion of fish (Mines et al 1997). However, statistics on its incidence
do not exist. Many countries do not have adequate systems for reporting foodborne
diseases. In those countries that do, cases are missed because of the often mild nature
of the illness, which causes patients not to seek medical attention, or ignorance by
medical personnel, who may misdiagnose it as a food allergy. Moreover, scombroid
poisoning is not a reportable disease, even in those countries that keep records (Taylor
1986). Under reporting of this illness is a worldwide problem (FDA 1992).

Since 1970, the countries with the most reported incidents of HFP are Japan, the US
and Great Britain, although this most likely represents better reporting by these
countries. Less frequent outbreaks have been reported in various other countries
32
including Canada, New Zealand, France, Germany, Norway, Sweden,
Czechoslovakia, the Netherlands, Australia, Sri Lanka, Indonesia, South Africa and
Egypt. Since only a few of these countries keep official records on incidents of fish
poisoning, it would be safe to assume that many incidents in these countries are not
reported (Taylor 1986).

3.2.2 Africa

Although no information was found on the incidence of HFP in Africa, Ababouch et
al (1986) discussed histamine levels in commercially processed fish in Morocco,
indicating that the problem has existed there. Morocco has a large fisheries industry
based in the Atlantic Ocean, with sardines the predominant fish landed. Fishmeal is
the main product, followed by canned sardines, tuna and mackerel and fresh or frozen
fish. In 1986, worldwide sales of Moroccan fish products were falling, owing to
complaints about the quality of canned products and several outbreaks of food
poisoning in Europe implicating Moroccan products. In a survey of 248 samples of
commercially processed fish, histamine concentrations ranged from <0.01 to
694 mg/100 g. Ten samples (4%) had toxicologically significant levels of
>50 mg/100 g. Tuna showed the highest percentage of samples (7%) with histamine
levels >50 mg/100g.

Morocco has since developed a progressive strategy to meet European standards for
fish products, including the use of mandatory in-plant Hazard Analysis and Critical
Control Point (HACCP)-based quality control systems (Ababouch 1997).

3.2.3 Asia

HFP was recognised as a major cause of foodborne disease in Japan in the early
1950s, and remains a major foodborne disease in that country. The first reported
incident involved 700 people, which remains one of the largest outbreaks on record.
Forty-two outbreaks involving 4122 cases were reported by the Ministry of Health and
Welfare, Japan, in 197080. Incriminated fish included mackerel, tuna, anchovies,
sardines and marlin. The largest outbreak yet recorded in the world, involving 2656
cases, occurred in Japan in 1973 from the consumption of dried horse mackerel
(Trachurus japonicus). Given the Japanese preference for raw fish, it is surprising that
cooked fish have been involved in more incidents than raw fish. The utilisation of
only the highest quality fish in the raw fish market of Japan is probably the reason for
this (Taylor 1986).

HFP probably occurs frequently in Asia. High levels of histamine in fish on sale in
Asian countries have been reported in various FAO Fisheries reports (references cited
by Mitchell 1993). There are also reports of extremely high levels of histamine in
some salted, and dried or salted, fermented products in Asia. Histamine in canned
products from Asia has also been a problem (James and Olley 1985).

3.2.4 Australia

There is little in the scientific literature on outbreaks of HFP in Australia, which
indicates that the disease is not common in this country. Incidents have been reported
33
associated with the consumption of tailor (Pomatomus saltatrix) (Taylor 1985),
juvenile Australian salmon (Arripis truttaceus) (Smart 1992) and tuna of unknown
source (Brown 1993) (see below). The present authors are unaware of any recent
media items on HFP. Information on surveys and monitoring presented here under
Histamine levels in fish products in Australia is reassuring, even if histamine is not
the only toxin involved, and indicates that HFP is unlikely to be prevalent in
Australia, at least in commercial fish products.

3.2.4.1 Outbreaks described by Smart (1992)

Smart (1992) reported two separate outbreaks of HFP caused by consumption of
juvenile Australian salmon (Arripis truttaceus), or salmon trout, caught in South
Australian waters. The fish had been purchased from two different local Adelaide fish
suppliers.

In the first outbreak, in March 1990, a family consisting of mother, child and infant
was affected. The fish was frozen within 1 h of purchase. Two weeks later, it was
washed and baked after partial defrosting at room temperature for 23 h and then in a
microwave oven. The mother, who noted that the fish had a peppery taste, required
parenteral promethazine (an antihistamine drug) and overnight hospitalisation.

The second outbreak, in March 1991, involved a family of three adults and one child.
The fish was frozen within half an hour of purchase. Before baking 1 week later, it
was defrosted in a refrigerator for 12 h. A 35-year-old man and a 69-year-old woman
required parenteral antihistamines, but were not hospitalised. Another woman and an
8-year-old boy did not require treatment.

In all cases, onset of symptoms occurred within half an hour of fish ingestion. They
included erythema and urticaria of the skin, facial flushing and sweating, palpitations,
hot flushes, headache, nausea, vomiting and dizziness. The patients in each outbreak
had minimal gastrointestinal symptoms. The infant, who consumed only one
teaspoonful of fish, was least affected of the seven patients, exhibiting only a transient
erythematous rash.

The following table summarises the clinical signs and symptoms in the seven patients
involved in the two outbreaks described by Smart (1992).

Signs and symptoms associated with HFP
in seven patients (Smart 1992)

Sign or symptom No. of patients
Erythema and urticaria
Facial flushing/sweating
Palpitations
Hot flush of body
Headache
Nausea
Vomiting
Dizziness
Peppery taste
7
5
5
3
3
2
1
1
1

34
The diagnosis of scombroid poisoning was made both clinically and on the
demonstration of high histamine levels in the cooked fish (80 mg/100 g and
254 mg/100 g in the first and second outbreaks respectively). This indicates gross
temperature abuse.

3.2.4.2 Cases described by Brown (1993)

Brown (1993) reported two cases of HFP in patients attending a suburban general
practice in Brisbane on one afternoon. The first patient was a 35-year-old nurse, who
presented with a florid flush of her entire skin surface. She complained of a pounding
heart, tightness of the chest and a feeling of doom. She had eaten tuna at a restaurant
2 hours before. Her symptoms had begun about 75 min before presentation and
steadily increased in intensity, in association with an extreme sense of alarm. She had
no itch, nausea or respiratory compromise. The woman was treated with 50 mg
promethazine by intramuscular injection. After 15 min, the erythema began to wane,
disappearing over the next 10 min. The patient said she felt much better. After a
further 10 min, loose bowel motions began, and continued intermittently for the next
45 min.

The second patient reported by Brown (1993) was a 31-year-old man, who had eaten
the same tuna dish. His symptoms were a hot sensation, nausea, diarrhoea, shakes and
headache. A third person who had eaten the dish was reported as having similar, but
less intense, symptoms.

3.2.5 Canada

In Canada, HFP was first confirmed in 1975, although there were probably incidents
before then. In the 1970s and 1980s, canned tuna was the most likely vehicle of
contamination, but between 1990 and 1995 it was fresh tuna, mahi-mahi or marlin.
The three largest outbreaks were caused by the ingestion of smoked mackerel
(October 1987: 14 cases), fresh marlin (July 1991: 12 cases) and fresh tuna (August
1994: 12 cases). Histamine levels of 28710 mg/100 g were implicated in seven
separate incidents involving tuna imported from Sri Lanka. Most incidents in Canada
have been associated with imported fish (Todd et al 1992; Todd 1997).

3.2.6 Europe

An early outbreak of HFP in Great Britain occurred among a group of British sailors
aboard the Triton of Leith in 1828 (Henderson 1830). Five crew members became ill
with symptoms of severe headache, dilated blood vessels, oedema, facial swelling and
flushing, swelling of the entire body and shivering following the consumption of
bonito. Bonito is a scombroid fish and a likely vehicle for HFP. After this early
incident, reports of HFP in Great Britain were scarce or non-existent until 1976. From
1976, numerous outbreaks have been reported in England, Wales and Scotland,
associated with mackerel, tuna, sardines, herring and pilchards (e.g. Gilbert et al 1980;
Bartholomew et al 1987).

Scoging (1991) reported on incidents of scombrotoxic fish poisoning in the United
Kingdom between 1976 and 1990. There were 441 suspected incidents involving 962
35
cases. Fish associated with 167 incidents were confirmed as having histamine levels
of >5 mg/100 g.

Between 1987 and 1996, fresh tuna and canned tuna became increasingly associated
with incidents of HFP. Scoging (1998) investigated 405 incidents during this period:
243 sporadic incidents (60%), 105 general outbreaks (26%) and 56 family outbreaks
(14%). In 148/405 incidents (37%), food remnants contained histamine at levels
>5 mg/100 g, and 117/148 (79%) had histamine levels >100 mg/100 g. (Histamine
was detected using HPLC.)

Of the 257 incidents not confirmed by elevated histamine levels, 61% (156) were
associated with atypical symptoms or with fish not commonly associated with this
syndrome (e.g. shellfish, crustaceans, trout, haddock, skate, shark), or directly
implicated remnants were unavailable for analysis. In the remaining 101 (39%),
symptoms and onset times were typical, but the levels of histamine found in remnants
were within acceptable limits. The latter would suggest uneven distribution of
histamine in contaminated fish or that unidentified agents or toxins may contribute to
the syndrome (Scoging 1998).

Tuna (fresh/frozen and canned) and mackerel were most commonly implicated in both
suspected and confirmed (elevated histamine) scombrotoxic incidents. Salmon was
involved in 30 suspected incidents, but remnants from only one incident contained
slightly elevated histamine levels (5.1 mg/100 g). Salmon is therefore of particular
interest as a vehicle for other possible causative toxins. Bacterial contamination
occurred both during harvesting and processing and during food retail and preparation
(e.g. after canned tuna was opened) (Scoging 1998).

Sockett (1991) reported on food poisoning outbreaks associated with manufactured
foods in England and Wales between 1980 and 1989. Thirty-five outbreaks associated
with processed fish and shellfish were reported during this period, including two
outbreaks caused by imported products. Scombrotoxin poisoning was the most
common cause of illness, and gave rise to 20 outbreaks affecting 59 people. Each
outbreak affected two or three people. All outbreaks were associated with mackerel
products, including smoked mackerel (18), mackerel pat (1) and filleted mackerel
(1). The symptoms were those of histamine poisoning.

Molinari et al (1989) reviewed the hygiene and health importance of histamine
toxicity in Italy and reported that the problem is widely underestimated in that
country. Tuna and mackerel were the fish most commonly associated with toxicity.
An outbreak of HFP occurred in Palermo in 1979 that affected 'nearly 250' people.

In 25 years (196691), 76 poisoning incidents after intake of tuna were reported to the
Swiss Toxicological Information Centre (Maire et al 1992). Twenty-seven reports
came from physicians and, of these, 18 fulfilled the criteria of scombroid fish
poisoning.

36
3.2.7 New Zealand

From 1973 to 1975, several incidents of histamine poisoning occurred in New
Zealand. The outbreaks were associated with consumption of canned mackerel,
smoked kahawai, kingfish and trumpeter fish. In addition, New Zealand authorities
assisted in the investigation of an outbreak in 1974 involving canned skipjack tuna in
the Solomon Islands (Taylor 1985).

In a report on HFP prepared in 1993 for the New Zealand Ministry of Health (Mitchell
1993), survey data from 199293 on histamine levels in (mostly smoked) fish were
presented, together with a summary of recent (199093) HFP outbreaks in New
Zealand. Nineteen outbreaks were reported between March 1990 and June 1993, all
resulting from the consumption of smoked fish, including kahawai and mackerel.
Histamine levels in the incriminated fish ranged from 20 to 500 mg/100 g. Mitchell's
(1993) report concluded that, because of the number of poisoning incidents and the
number of samples exceeding the acceptable level of histamine in retail smoked fish,
further monitoring and investigations of the causes of high levels of histamine in fish
were warranted.

3.2.8 United States

It is difficult to determine the true incidence of HFP in the US, because of incomplete
record keeping. Local health departments recommend, but do not require, that cases
be reported. Statistics on the disease are also inaccurate because of misdiagnosis
(Lange 1988). Data compiled by the CDC in the mid-1970s indicated no particular
season for HFP and suggested Hawaii and California as the states with largest number
of outbreaks (Hughes et al 1977). In 1980, histamine poisoning was one of the most
prevalent forms of foodborne disease of chemical aetiology in the United States,
ranking only behind ciguatera fish poisoning (Sours and Smith 1980). By 1997,
scombroid toxicity or HFP was the most prevalent form of seafood-borne disease in
the United States Lipp and Rose (1997) reported outbreaks of seafood-borne
disease associated with fish, 198392, and listed the aetiological agents as scombroid
(57% of outbreaks), ciguatoxin (19%), bacteria (14%), unknown (9%) and chemicals
(1%). In contrast to the Japanese situation, most of the US outbreaks have involved a
small number of people, typically fewer than five individuals.

Between 1970 and 1974, 'scombroid fish poisoning' was responsible for 29 (43%) of
the 68 outbreaks caused by fish and shellfish toxins. In 45% of outbreaks, laboratory
tests demonstrated elevated histamine levels in the incriminated fish. Tuna and mahi-
mahi were most frequently implicated. The place where fish were mishandled, leading
to the outbreak, was identified in 10 outbreaks. Errors leading to contamination of the
food were reported in 18 outbreaks, and improper storage or holding temperature was
identified in 16 (89%) of these outbreaks (Hughes et al 1977).

In 1970, some 40 children in a school lunch program became ill after eating imported
canned tuna. A larger outbreak of HFP occurred in 1973, involving commercially
canned tuna (Merson et al 1974). There were 254 clinical cases in eight states. The
symptoms included immediate oral burning and blistering, followed in 3045 min by
headache, abdominal cramps, diarrhoea and flushing. No cases required
37
hospitalisation, and the duration of illness was generally 8 h or less. Nine assays for
histamine produced values ranging from 68 to 280 mg/100 g, with controls at
3 mg/100 g. FDA recalled the incriminated lots, which showed evidence of
honeycombing, indicating advanced decomposition (Merson et al 1974; Arnold and
Brown 1978). This outbreak is probably still the largest on record in the United States,
and it signalled an increasing awareness of histamine poisoning in that country
(Taylor 1986). In 197980, more than 200 people became ill after consuming
imported frozen mahi-mahi.

Other incidents of intoxication in the United States have resulted from the
consumption of canned abalone-like products, canned anchovies, and fresh and frozen
amberjack, bluefish (tailor), sole and scallops. In particular, shipments of unfrozen
fish packed in refrigerated containers have posed a significant problem because of
inadequate temperature control (FDA 1999).

3.3 Fish characteristics that affect the clinical response

3.3.1 Species

Fish that cause HFP include mackerel (Scomber spp.), tuna (Thunnus spp.), saury
(Cololabis saira) and bonito (Sarda app.) (scombroid fish); and mahi-mahi
(Coryphaena spp.), sardines (Sardinella spp.), pilchards (Sardina pilchardus),
anchovies (Engraulis spp.), herring (Clupea spp.), marlin (Makaira spp.) and bluefish
(Pomatomus spp.) (non-scombroid fish) (Taylor 1986; Institute of Medicine 1991).
Non-scombroid species Australian salmon (Arripis truttaceus), sockeye salmon
(Oncorhynchus nerka) and Cape yellowtail (Seriola lalandii) have also been
implicated in HFP (Smart 1992; Mller et al 1992; Gessner et al 1996). As non-
scombroid fish can cause HFP, the term scombroid fish poisoning is a misnomer
(Taylor 1988).

In the USA, of 73 outbreaks of HFP reported to the Centers for Disease Control from
1978 to 1982, 42% implicated mahi-mahi as the vehicle. An epidemic in Alabama and
Tennessee in 1986 involved amberjack (Seriola spp.), a non-scombroid species.
Bluefish have been responsible for many cases of HFP along the eastern seaboard of
the US (Karolus et al 1985; Etkind et al 1987; Lange 1988).

In New Zealand, incidents involving smoked kahawai, kingfish and canned mackerel
in the early 1970s were reported and discussed by Foo (1975a,b, 1976, 1977). Other
reports involved kingfish and smoked kahawai (Mitchell 1984) and smoked mackerel
(Mitchell and O'Brien 1992). Mitchell (1993) gave a summary of recent cases
involving smoked kahawai, smoked marlin, smoked mackerel and smoked trevally.

Australian cases were caused by improperly stored tailor or bluefish (Pomatomus
saltatrix) (Taylor 1985); juvenile Australian salmon (Arripis truttaceus) (Smart 1992);
and tuna (Brown 1993).

38
3.3.2 Intrinsic potential for histamine and cadaverine accumulation

Since free histidine in fish muscle is the substrate for microbial decarboxylation to
produce histamine, species difference in histidine content has a large effect on the
potential hazards of poor handling practices (Pan 1985b). Although the muscle of
fatty, red-meat, active and migratory species contains much more free histidine than
that of white-meat, slower species, the latter still contain combined histidine as a
component of muscle protein. In the active fish, the free amino acid may act as a
buffer, protecting the tissues from the effects of sudden increases in lactic acid.
Histidine deaminase [HAL] and urocanase, which mediate the first two steps of the
degradative process, regulate the level of histidine in the live fish (Love 1980).

Arnold and Brown (1978) cited a series of papers dealing with the freshness of fish
and the amount of histamine present. They discussed factors such as the effect of pH
and temperature on the amount of histamine produced in red-meat fish such as
mackerel and white-meat fish such as rockfish. The terms red-meat fish and white-
meat fish are based on the general surface appearance of the fish. The terms red
muscle and white muscle refer to muscle types within individual fish, red muscles
being those containing predominantly red fibre types and white muscles being those
containing predominantly white fibre types. All scombroid fish contain both muscle
types in varying amounts, depending on species. In the commercial processing of tuna,
only white muscle is canned for human consumption, the red muscle being used
primarily for pet food (Arnold and Brown 1978).

Takagi et al (1969) examined the amounts of histidine and histamine in 21 species of
aquatic animals during spoilage and found that more histamine was produced in red-
meat fish such as mackerel than in white-meat fish such as rockfish. Suyama and
Yoshizawa (1973) found relatively high free histidine contents in 13 species of
migratory red-meat fish (2861460 mg/100 g), whereas white-meat fish were low in
free histidine (038.2 mg/100 g) and did not produce histamine during spoilage.
However, within the same species of fish, more histidine was found in white than in
red muscle, and the resulting histamine formation followed this pattern with regard to
muscle type; i.e. more histamine in white muscle (Takagi et al 1969; Orejana et al
1983; Pan 1985b). The following table illustrates the results of random sampling of
fish from a fish market for histamine content of red and white muscle.

Histamine content in the white and red muscle of scombroid fish
(after Pan 1985b)

Histamine (ppm) Common name Scientific name
White muscle Red muscle
Spotted mackerel
Frigate mackerel
Oceanic bonito
Scomber australasicus
Auxis thazard
Euthynnus affinis
25
45
80
3
6
35

Klausen and Lund (1986) set up an experiment to demonstrate the differences in the
amine formation between a scombroid and a non-scombroid fish. They chose
mackerel (Scomber scombrus) and herring (Clupea harengus), because in Denmark
the methods of catching, handling and manufacturing of the two species are very
much alike, but only mackerel causes HFP. The fish were stored in refrigerated tanks
39
or ice until they arrived at the laboratory and were eviscerated, vacuum packed and
stored. When stored in vacuum packs at 2 or 10
o
C, mackerel and herring accumulated
similar amounts of histamine (herring, 4.2 and 10.9 mg/100 g; mackerel, 4.3 and
11.4 mg/100 g respectively) at the time of rejection, which was assessed by sensory
means by a panel of six.

With cadaverine, the situation was different. In herring, only small amounts were
formed (0.7 mg/100 g at 2
o
C and 4.9 mg/100 g at 10
o
C). In mackerel, high contents of
cadaverine rapidly accumulated 20 mg/100 g at 2
o
C and 22.6 mg/100 g at 10
o
C
(before rejection). These values for mackerel correspond to about 25 times the
histamine contents. Thus the levels of cadaverine were high enough in relation to
histamine to potentiate the oral toxicity of histamine as described in guinea pigs by
Bjeldanes et al (1978), and may explain why mackerel are much more frequently
involved than herring in HFP incidents.

In addition, according to Klausen and Lund (1986) the contents of free histidine and
free lysine are 45 times higher in fresh mackerel compared with fresh herring. When
exposed to high temperatures (2030
o
C) and contamination with histidine-
decarboxylating bacteria, mackerel therefore has the capacity for accumulation of
much higher levels of histamine and cadaverine than herring. Gilbert et al (1980)
examined 37 samples (almost all of them mackerel) involved in HFP and found
>100 mg/100 g of histamine in 22/37. In herring, on the other hand, only moderately
toxic levels of histamine (50 mg/100 g) could be formed from the free histidine
present in the fresh fish (Klausen and Lund 1986).

Cadaverine is more frequently found in spoiled fish than is histamine. The amount
varies, but levels of 1060 mg/100 g have often been found. Putrescine levels in
spoiled fish are usually much lower than those of cadaverine, usually <10 mg/100 g,
probably because of the limited quantities of ornithine in fish tissues (Taylor and
Sumner 1986).

While the significance of cadaverine and putrescine as potentiators of histamine
intoxication has not been fully established, it could be expected that if they are present
at ratios to histamine higher than those normally found in spoiled fish, they may
inhibit the detoxification of histamine. They may also act in an additive or synergistic
fashion with other, as yet undiscovered, potentiators.

3.3.3 Seasonal and other variability

Whereas data compiled by the CDC in the mid-1970s indicated no particular season
for HFP (cited by Lange 1988), there seems to be a definite connection between
season, temperature, histidine content and histamine formation in fish products (Pan
1985b). Free histidine content in herring varied with seasons from 260 to 1600 mg/kg,
being highest in summer (Hughes 1959). Less histamine was found in sardines and
bonito during winter than in other seasons under the same handling conditions
(Simidu and Hibiki 1954). Skovgaard and Elleman (1978) reported that confirmed
histamine poisoning outbreaks caused by smoked mackerel occurred during late
summer, when both fish and water have the highest temperature, and the mackerel are
at their fattest.
40

Clifford et al (1989) noted that mackerel show quite marked fish-to-fish and seasonal
variations in chemical composition and susceptibility to spoilage by various
microbiological, enzymic and auto-oxidative processes. The authors concluded that it
is possible that only certain fish in an apparently homogenous batch will develop all
factors necessary to induce HFP.

3.3.4 Parts of fish consumed

Concentrations of histamine in fish tend to vary widely from one part of the fish to
another. Histamine in raw fish is usually present at higher levels in tissue adjacent to
the gills or the intestines, the main reservoirs of the histidine decarboxylating bacteria
(HDB) (Lerke et al 1978; Taylor 1986). Frank et al (1981) observed a histamine
gradient in spoiled skipjack tuna, with highest concentrations near the gut cavity in the
anterior section, and progressively lower concentrations moving posteriorly. Orejana
et al (1983) found that during 15-day iced storage of skipjack tuna, the tail section
showed the lowest histamine content throughout the period. Hardy and Smith (1976)
showed that the histamine content of ungutted mackerel was 10 times more than that
of gutted fish after storage at ambient temperature for 140 h.

3.3.5 Nature and amount of bacterial contamination in spoiling fish

3.3.5.1 The common histamine-producing bacteria

Although only about 1% of the surface microflora of live fish represents histamine
producers (Kimata 1961), HDB form a greater proportion of the microbial population
as a fish spoils. Omura et al (1978) reported that 31% of isolates from spoiled
skipjack tuna and jack mackerel growing at warm temperatures were histamine
producers, and Yoshinaga and Frank (1982) found 13.4% in decomposing skipjack
tuna. Frank and Baranowski (1984) found with mahi-mahi that 7% of isolates growing
at warm temperatures and 9% growing at refrigeration temperatures were histamine
producers.

Taylor et al (1978a) identified 112 species of bacteria that are known to possess HD.
Most were members of the family Enterobacteriaceae, or the genera Clostridium and
Lactobacillus. Enteric bacteria, specifically Morganella morganii, certain strains of
Klebsiella pneumoniae and a few strains of Hafnia alvei are the most prolific
histamine producers in fish when they are maintained at temperatures greater than 4
o
C
(Stratton and Taylor 1991) and are the most commonly associated with scombrotoxic
fish (Taylor 1983). Certain strains of Lactobacillus that are also prolific histamine
producers are probably of importance only in fermented fish (Taylor 1986).

Niven et al (1981) identified Vibrio spp. as histamine producers, and histamine-
producing isolates of V. alginolyticus have been found in decomposing skipjack tuna
and mahi-mahi (Frank et al 1985). V. harvei, V. fisheri and Photobacterium leiognathi
are capable of producing histamine at warm temperatures (Ramesh and Venugopalan
1986). Other Photobacterium spp. and Vibrio spp. may be primarily responsible for
histamine production in scombroid fish at lower temperatures (Morii et al 1986; Van
Spreekens 1987; Leung 1987).
41

3.3.5.2 Temperaturegrowth relationships and histamine production

Doe et al (1998) pointed out that the traditional classification of bacteria into
psychrophiles, psychrotrophs, mesophiles and thermophiles was only arbitrary and
that there is a gradation from one category to the other. Because of the overlapping in
growth curve characteristics and inconsistencies in the use of these categories in the
literature, the traditional classification will not be used in this review. Rather, the
reader is asked to utilise the concept displayed in Figure 3 below from McMeekin et
al (1993), where the square root of the bacterial growth rate is plotted against
temperature for organisms typical of four thermal classes, groups A, B, C and D.

0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Temperature (C)
1
/
(
g
e
n
e
r
a
t
i
o
n

t
i
m
e
,
[
h
]
)
A B C D
Figure 3. Growth characteristics for four categories of bacteria at temperatures used in
fish processing and storage (adapted from Fig. 10.7 of McMeekin et al 1993).
Common histamine producers can be found across much of the temperaturegrowth
spectrum: Photobacterium phosphoreum (group A); several Vibrio species (group B);
Klebsiella pneumoniae, Morganella morganii and Hafnia alvei (group C); and group
D bacteria, if present.

The temperatures at which the growth curves peak represent the temperatures at which
growth is optimal for four organisms representative of each of the four recognised
thermal classes. These temperatures are equivalent to:

1. Maximum refrigeration temperature (5
o
C)
2. Temperate ambient temperature (20
o
C) and temperatures used for cold
smoking in temperate areas (<30
o
C)
3. Tropical ambient temperature and temperature used for cold smoking in
tropical areas (40
o
C)
4. Temperature in a hot smoking kiln (60
o
C).

From Figure 3 it can be seen that at refrigeration temperatures bacteria in group A are
nearing their maximum growth rate and group B are growing slowly. At temperate
ambient temperature and during cold smoking in temperate area (Anon. 1988a), the
growth of group A has slowed markedly, that of B is close to the maximum and C is
42
growing slowly. At tropical ambient temperature and temperatures used for cold
smoking in tropical areas (Chng and Kuang 1987), group C is almost at its optimum
growth rate, B is almost at its upper limit of temperature, and D is beginning to grow
well. In temperatures in the hot smoking kiln (Anon. 1988b), bacteria in groups A, B
and C have been eliminated, but 60
o
C has not destroyed group-D bacteria,
representing the thermophile group, if there are any present.

Each bacterial strain has a notional temperature (Tmin) at which growth is zero on the
temperature axis; a temperature at which growth is most rapid (Topt); and a
temperature at which thermal death reduces the growth rate to zero (Tmax). Cardinal
temperatures predicted from data for a wide range of organisms were listed by
McMeekin et al (1993). From these data, it is evident that there are no clear-cut
distinctions between the temperature groups, but rather a continuum of Tmin values. It
is interesting to note the lower limits for production of toxicologically significant
levels of histamine in tuna fish infusion broth for some common HDB were 7
o
C for K.
pneumoniae, 15
o
C for two M. morganii strains, and 30
o
C for H. alvei, Citrobacter
freundii and E. coli (Behling and Taylor 1982).

Because of the importance of temperature in the production of histamine, several
attempts have been made to predict histamine formation in spoiling fish at different
temperatures. For example, Frank (1985) constructed normographs to predict
histamine production in skipjack tuna, and Pan (1985a) used Arrhenius plots for such
estimations in mackerel and bonito. However, the methods involved too many
assumptions and were inaccurate in their predictions. A further development,
temperature function integrators, which take temperature fluctuations into account,
were also considered unsuitable for predicting the complicated process of histamine
production in spoiling fish (Olley and McMeekin 1985).

It is clearly not possible to relate the amount of bacterial contamination to histamine
production by such methods. Numerous factors introduce bias. First, a reasonable
estimate of the initial numbers of significant bacteria needs to be made. Then
histamine concentration represents the amount produced by potentially diverse
population of bacteria with different capacities for histamine production and/or
destruction in association with bacteria that are not histamine producers or destroyers.
Even when one group or strain of HDB predominates, histamine production is
unlikely to follow a growth curve fitting that group/strain, not least because
metabolism of histamine by histaminase may be taking place simultaneously. Further,
the amount of histamine production varies from site to site within a fish, and substrate
changes such as proteolysis at high temperatures affect the amount of histidine
available for histamine formation.

Subsequent cooking or processing of a spoiled fish will further alter the relationship
between bacterial numbers and histamine production by reducing or removing the
microbial population without affecting the histamine content significantly. For
example, Fletcher et al (1995) reported that, when whole fresh kahawai (Arripis
trutta) was stored under a variety of temperature regimes, elevated levels of histamine
(defined as 50 mg/kg) occurred only when aerobic plate counts at 20 or 35
o
C
exceeded 10
6
colony forming units (CFU)/g. There was only one sample with elevated
histamine where the aerobic plate count was <10
7
CFU/g. When such fish were
43
smoked, bacterial numbers, but not histamine concentration and toxicity, were
considerably reduced (Fletcher et al 1998).

3.3.5.3 Studies on variability in species/strain composition of microflora

Scientists have found that factors affecting the dominating species/strains of HDB in
the microflora of a particular spoiling fish are many and varied. The diversity can be
attributed to differences in the species of fish, handling procedures, holding times and
temperatures. In addition, the character of the microflora can be influenced by the
fishs feeding habits, geographical location, the season, ocean temperature etc.
(Kimata 1961; Shewan 1977; Yoshinaga and Frank 1982). It is not surprising that
results have been variable in many studies on the effect of storage temperature of
histamine formation in fish (Behling and Taylor 1982).

HDB that grow at refrigeration temperatures usually produce histamine in smaller
quantities than those produced by species that grow at warmer temperatures, and toxic
levels of histamine may not be reached. Okuzumi et al (1984a) looked at the
occurrence of various histamine-forming bacteria on/in fresh fish purchased from fish
markets in Japan. For the summer samples, M. morganii was found in the greatest
numbers, followed by the N-group bacteria, later identified as Photobacterium
phosphoreum by Fujii et al (1997). For the winter samples, only N-group bacteria
were detected. This fits in with the observation of Simidu and Hibiki (1954), who
found less histamine in sardines and bonito during winter than in other seasons under
the same handling conditions.

In a subsequent study, Okuzumi et al (1984b) studied microbial populations at various
storage temperatures in mackerel bought at fish markets, and correlated these with
histamine production. In fish samples stored at 5
o
C and 10
o
C, the main histamine
formers were N-group bacteria, which reached levels of 10
7
10
8
/g after 7 days. In
samples stored at 15
o
C, the formation of histamine was still attributed mainly to N-
group bacteria, particularly in the first 3 days, although numbers of M. morganii
increased throughout. In samples stored at 20
o
C, a great part of histamine at the early
part of storage (04 days) was attributed to N-group bacteria, and at a later stage (6
8 days) to M. morganii. However, at the final stage (1114 days), histamine content
decreased from >700 mg/100 g, and remained constant at about 500 mg/100 g. In
samples stored at 25
o
C and 30
o
C, M. morganii was the main histamine producer.

Ryser et al (1984) obtained 60 isolates of indigenous bacteria from raw tuna fish, and
identified them as Pseudomonas spp. Fewer than half (35%) the isolates produced
histamine during incubation at 21
o
C for 48 h, and the maximum amount produced by
a single isolate was 3.2 mg/100 mL, far less than the 50 mg/100 g believed necessary
to induce symptoms of HFP. This would suggest that bacteria that grow at lower
temperatures are not significant toxicologically. However, Okuzumi et al (1981)
reported significant histamine production by halophilic (salt-loving) N-group bacteria
growing at 5
o
C (300400 mg histamine/100 mL of mackerel homogenate).

Husain (1996) estimated the growth rates of different HDB such as Vibrio spp.,
Bacillus spp. and Morganella spp. incubated at 28
o
C in seawater broth (75% sterile
44
seawater, 0.5% peptone and 0.5% yeast extract) at different salt concentrations. At
sodium chloride concentrations ranging from 1% to 10%, a visible growth was seen in
all cultures. Maximum growth occurred at 4%, 3% and 7% sodium chloride for Vibrio
spp., Bacillus spp. and Morganella spp. respectively.

Husain (1996) also found that traditional Indian preservatives garlic, turmeric, ginger
and black pepper all had a profound inhibitory effect on the growth of HDB isolates at
concentrations of 1% to 5%. There was a large decrease in bacterial growth rate as
concentrations increased. Garlic was the most effective, completely inhibiting growth
of Vibrio spp. at 4% concentration and Bacillus spp. and Morganella spp. at 5%
concentration.

Earlier, workers in Japan (Wendakoon and Sakaguchi, 1992) had tested the effects of
powdered spices and their ethanol extracts on growth and biogenic amine formation of
Enterobacter aerogenes and M. morganii. Clove and cinnamon were the best
inhibitors of the spices tested (allspice, cardamom, chilli, cinnamon, clove, cumin,
black pepper, nutmeg, sage and thyme).

3.3.5.4 Species/strain variation in histidine decarboxylase activity

Different bacteria vary significantly in the quantity of histidine decarboxylase (HD)
they produce and/or the specific activity of their decarboxylases. According to
Ferencik (1970), M. morganii (then P. morganii) required histidine levels of 100
200 mg/100 g to induce the production of HD. By contrast, H. alvei decarboxylated
histidine when it was present at <50 mg/100 g. This suggests that bacteria differ
according to their relative importance as histamine producers on different species of
fish.

Eitenmiller et al (1981, 1982) investigated factors influencing HD production by M.
(then Proteus) morganii. Examination of 22 strains of M. morganii revealed that each
possessed HD activity, although at variable levels. With one high-activity strain
(GRMO 6), maximal HD activity occurred at 37
o
C and pH 6.5. Normal muscle pH of
fresh yellowfin tuna (6.5) thus corresponds closely to the pH required for optimal
activity of HD, and is low enough to permit rapid enzyme synthesis by M. morganii.
Minimal enzyme activity was present when the culture was grown at pH 8.5. HD
activity decreased as the age of the culture increased. Rapid enzyme and histamine
formation occurred in yellowfin tuna fillets inoculated with M. morganii and stored at
24 and 30
o
C. Histamine soon reached toxic levels of 520 mg and 608 mg/100 g at 24
and 30
o
C respectively. Little enzyme activity was present in inoculated fillets stored at
15
o
C, and in control fillets that were not inoculated.

Olley and Baranowski (1985) pointed out that low-temperature enzyme activity by
microorganisms that grow at warm temperatures is important in histamine formation,
provided sufficient bacterial numbers have been reached before cold storage. Klausen
and Huss (1987b) studied growth and histamine production by M. morganii in
histidine-containing broth and in mackerel. Following storage at higher temperatures
(1025
o
C), large amounts of histamine were formed at low temperatures (05
o
C),
when no growth took place. Fujii et al (1994) found that the specific activity of HD of
halophilic histamine-forming bacteria Photobacterium phosphoreum and P.
45
histaminum sp. nov. remained at 27% and 53% of the initial value after storage in cell
suspensions for 7 days at 4
o
C and 20
o
C respectively, while the viable cells decreased
by 5 log cycles and 8 log cycles of the initial counts respectively. This indicates that
outbreaks of HFP may be caused by the ingestion of frozenthawed fish even when
the viable count of histamine forming bacteria is low.

A common feature of studies on bacteria producing HD is that when multiple strains
of the same species are isolated, only one or a few of these strains are prolific
histamine producers (Gale 1946; Voigt and Eitenmiller 1977; Taylor et al 1978a;
Taylor et al 1979; Yoshinaga and Frank 1982). Most strains of M. morganii are able
to produce histamine, but only certain strains of K. pneumoniae and H. alvei are
described as potent histamine producers (Havelka 1967; Taylor et al 1978a; Taylor et
al 1979). Taylor et al (1979) isolated a histamine-producing strain of K. pneumoniae
from a sample of tuna sashimi implicated in an outbreak of HFP. The strain was
capable of producing 442 mg of histamine per 100 g/tuna in tuna fish infusion broth in
7 h under controlled incubation conditions. Only 12/50 other K. pneumoniae strains
isolated from foods, representing five distinct biochemical types, were able to produce
such high levels of histamine in the broth.

Behling and Taylor (1982) divided HDB into those species capable of producing large
quantities of histamine (>100 mg/100 mL) in tuna fish infusion broth during a short
incubation period (<24 h) at temperatures >15
o
C and those capable of producing
somewhat lesser quantities (<25 mg/100 mL) after prolonged incubation (>48 h) at
30
o
C or above. M. morganii, K. pneumoniae and Enterobacter aerogenes were
prolific histamine producers, and tested strains of H. alvei, C. freundii and E. coli
were slow producers (Taylor et al 1978a; Behling and Taylor 1982). Taylor et al
(1978a) suggested that most of the other bacteria identified as histamine producers in
the scientific literature fall into the slow-producer category. However, in reporting that
M. morganii, K. pneumoniae and H. alvei were the only bacteria that had been
isolated from fish implicated in fish poisoning incidents, Taylor (1985) postulated that
certain other bacteria may also fall into the prolific-histamine-producer classification,
citing as an example the isolation of C. perfringens from decomposing skipjack tuna
by Yoshinaga and Frank (1982).

Leung (1987) found that, of 36 bacterial isolates from spoiled chub mackerel
(Rastrelliger sp.) skin washings, all the Morganella strains (10) produced large
quantities of histamine. However, of two strains of Acinetobacter calcoaceticus
isolated, one was the strongest producer of all strains tested whereas the other gave a
totally negative result. Similarly, only 1/3 K. pneumoniae strains and 1/4 Aeromonas
hydrophila strains were histamine producers. Thus, for the range of organisms
isolated in the study, apart from Morganella spp., the presence of HD was not a
consistent feature exhibited by species. In attempting to explain these results, Leung
(1987) postulated that HD may be controlled by a plasmid and that the plasmid may
be transferred from strain to strain, species to species or genus to genus. Subsequently
Barancin et al (1998) found that the fish pathogen Vibrio anguillarum harbours a
plasmid-encoded HD gene. Taylor et al (1979) found no correlation between
histamine production and other biochemical characteristics or antibiotic resistance,
which may also be linked to plasmid genes.

46
Many bacteria (especially of the Enterobacteriaceae family) possess either ornithine
decarboxylase or lysine decarboxylase, the enzymes necessary to produce potential
potentiators of histamine, putrescine and cadaverine, respectively (Taylor and Sumner
1986). Since only a few bacteria possess HD responsible for histamine production, it
is likely that the bacteria forming histamine differ from those forming putrescine and
cadaverine. Leung (1987) found that, of 36 bacterial isolates from spoiled chub
mackerel skin washings, none had the ability to decarboxylate all three amino acids,
histidine, lysine and ornithine.

3.3.5.5 Bacterial destruction of histamine

The histamine levels in a toxic fish depend on free histidine levels in the muscle and
the balance between histamine production and histamine destruction by the
contaminating microflora (Okuzumi et al 1984b). Histaminase (or DAO) activity has
been detected in several types of bacteria including M. morganii, Vibrio spp. and
Klebsiella spp. (Gale 1942; Satake et al 1953; Ienistea 1971). Gale (1942) found that
bacterial histaminase is best produced under somewhat alkaline conditions (pH 7.5
8), but that moderate activity also occurs under slightly acidic conditions. Yamanaka
(1984) showed that in saury, mackerel, yellowtail and big-eyed tuna the overall rate of
histamine production was greater at 20
o
C than at 35
o
C, while in skipjack tuna more
histamine was produced at 35
o
C. The lower levels of histamine production at high
temperatures in the former species was attributed either to histaminases or to HDB
with a growth optimum of <35
o
C (Olley and McMeekin 1985).

Ferencik (1970) reported that a strain of M. morganii, after being inoculated into a
sterile tuna flesh homogenate, produced large amounts of histamine. However, a
significant amount of histamine was soon decomposed by the organism. A subsequent
sterile addition of histidine to the inoculated homogenate again resulted in histamine
formation, followed by histamine destruction. He concluded that the histamine
production and destruction by the M. morganii strain was determined by the
concentration of free histidine in the fish homogenate.

3.3.6 Histamine levels and toxic dose

While the presence of histamine in fish muscle is a good indication that
decomposition has taken place, its occurrence is extremely variable. As is evident
from the above, its production is a function of the species of fish and the individual
fish, the part of the fish sampled, time, temperature, and the types and numbers of
bacteria present (Rawles et al 1996).

The threshold toxic dose for histamine in foods is not precisely known (Taylor 1986).
The variation in histamine levels within a spoiled fish (Lerke et al 1978; Frank et al
1981) makes estimates of the toxic threshold difficult to obtain. Lerke et al (1978)
showed that the distribution of histamine in spoiling tuna is quite uneven, varying
more than 4-fold over 3 cm, and being considerably higher near the gut cavity than
elsewhere. Yoshinaga and Frank (1982) attributed this to the non-uniform distribution
of spoilage organisms throughout the fish, and noted that muscle levels of histidine
were essentially uniform in fresh skipjack tuna. Simidu and Hibiki (1955) estimated
47
the threshold toxic dose for histamine in fish at about 60 mg/100 g, but their methods
were not precise.

Shalaby (1996) reviewed the oral toxicity to humans of histamine and other biogenic
amines in foods. He considered that histamine-induced poisoning is, in general, slight
at 840 mg, moderate at >40 mg and severe at >100 mg. Based on an analysis of
recent poisoning episodes, Shalaby (1996) suggested the following guideline levels
for histamine content of fish:

<5 mg/100 g (safe for consumption)
520 mg/100 g (possibly toxic)
20100 mg/100 g (probably toxic), and
>100 mg/100 g (toxic and unsafe for human consumption).
This assessment correlates with those of Simidu and Hibiki (1955) and Mitchell
(1993), that poisoning does occur at lower histamine concentrations than
100 mg/100 g fish.

There is uncertainty regarding the threshold toxic concentration because potentiators
of toxicity may be present in fish and lower the effective dosage compared with pure
histamine (Institute of Medicine 1991). Different fish could contain different
potentiators, and the levels of potentiators could also vary considerably from one fish
to another. The types and levels of potentiators in a fish would depend on a variety of
factors, including the natural constituents of the fish, the contaminating bacteria and
their metabolic capabilities, and the environmental conditions (mainly temperature).
Until the identity, levels and potency of possible potentiators and/or mast cell
degranulating factors are elucidated, it is difficult to establish regulatory limits for
histamine in foods on the basis of potential health hazard.

FDA guidelines for tuna, mahi-mahi and related fish specify 500 ppm (50 mg/100 g)
as the toxicity level, and 50 ppm (5 mg/100 g) as the defect action level because
histamine is not uniformly distributed in a decomposed fish. Therefore, if 50 ppm is
found in one section, there is a possibility that other units may exceed 500 ppm (FDA
1998). These levels, based on years of investigative experience, allow for the non-
uniform distribution of histamine in a spoiled fish, but ignore the existence of
suspected potentiators or other toxins.

For control of histamine in fish belonging to the Scombridae and Clupeidae families,
European Union Directive No. 91/493 stipulates that nine independent samples from
each batch should correspond to:

1. An average histamine concentration lower than 100 ppm (10 mg/100 g).
2. No more than 2 samples out of the 9 with a concentration of between 100 and
200 ppm.
3. No sample with a histamine content higher than 200 ppm.

The acceptable level set as a manufacturing standard by legislation in the United
Kingdom in 1992 was 10 mg/100 g (Anon. 1998b).

48
In Australia and New Zealand, the Australian Food Standards Code (Issue 41,
18 November 1998) Standard D1 Fish (ANZFA 1998a) states:

(1B)(a) The level of histamine in a composite sample of fish or fish products, other
than crustaceans and molluscs, when examined according to Section 977.13 of
Association of Official Analytical Chemists (AOAC) 15th Edition (1990) must not
exceed 100 mg/kg.

(b) For the purpose of this clause, a 'composite sample' is a sample, taken from each
lot, consisting of five portions of equal size taken from five representative samples.

This clause came into force in October 1994 (Peter Abbott, ANZFA, pers. comm.
1999) and seems conservative and appropriate to protect public health and safety.
However, it is currently under review, with a proposal by ANZFA to increase the
maximum allowable level of histamine in fish and fish products to 200 mg/kg
(ANZFA 1998b).

If we regard 10 mg histamine/100 g of fish as the highest level that can be consumed
safely, this must be related to the amount of fish eaten and the weight of the person to
calculate a likely safe dose. If a 60 kg person eats, say, 300 g (wet weight) of this fish,
this dose would be 0.5 mg/kg bw. Such a calculation is of limited value, however,
considering the variable nature of HFP and the lack of understanding of its
pathogenesis.

3.4 Human factors that affect the clinical response

3.4.1 Variation in individual susceptibility

The severity of the clinical response will depend on the amount of toxin(s) ingested
and variation in individual susceptibility. The literature would suggest that there is a
big variation in individual susceptibility. This was illustrated in the experiment of
Motil and Scrimshaw (1979) mentioned earlier. Oral administration of 100180 mg
histamine in grapefruit juice or in 100 g high-quality tuna caused characteristic
symptoms of mild histamine poisoning in only 1/4 and 4/8 volunteers respectively.

According to Taylor et al (1989) attack rates in group outbreaks of histamine
poisoning vary from 50% to 100%. In the human volunteer trials of Van Gelderen et
al (1992), only 2/8 subjects (both female) exhibited symptoms after ingesting portions
of spoiled fish containing 90 mg histamine, and these volunteers did not have higher
concentrations of histamine in plasma than the volunteers who did not show
symptoms.

In the four cases of 'classic scombrotoxism' described by Blakesley (1983), amounts
of histamine ingested were not known. Signs and symptoms varied between the
subjects. Three of four patients exhibited burning skin, headache and nausea, two of
four had abdominal cramps and pruritus, and one of four had hive-like skin lesions
and palpitations.

49
3.4.2 Influence of diet

Geiger (1955) suggested that alterations in the intestinal tract caused by seasoned hot
dishes prepared from spoiled fish or the simultaneous consumption of alcoholic
beverages might cause histamine to be absorbed at an increased rate, such that its
detoxification could not keep up with its entry into the circulation. Zee et al (1981)
also suggested that histamine may be absorbed more rapidly in the presence of alcohol
and exert a more-marked biological effect on the circulatory system. However, in the
rat, ethanol enhanced DAO and HMT activity to 123% and 111% of normal
respectively (Taylor and Lieber 1979). Mitchell and Code (1954) reported that
histamine taken with a meal (bread, butter and milk) was absorbed to a greater extent
than histamine consumed by itself.

Granerus (1968) also studied the effect of diet on histamine metabolism in humans.
His studies indicated the variability in urinary excretion of histamine and its main
metabolite, 1-methyl-4-imidazoleacetic acid. Granerus (1968) quoted a study by Irvine
et al (1959) that concluded that some intestinal bacteria could in fact contribute to
urinary histamine by decarboxylating histidine in food. While most proteins differ by
only 23% in their histidine content, these proteolytic bacteria are favoured by a diet
rich in animal protein and inhibited by a diet that favours the acidophilic group of
bacteria (e.g. vegetables, grains, lactose and dextrin). Thus, dietary differences may
explain the highly varying amounts of histamine metabolite found in the urine of some
subjects studied by Granerus (1968).

Foo (1977) cited an observation of Johnson and Overholt (1967) of a significant
increase in the concentration of histamine in the gastric venous blood of dogs whose
stomachs had been filled with dilute acetic acid solutions. He suggested that the lemon
or vinegar commonly used to enhance the flavour of fish might also enhance the
absorption of histamine from toxic fish. The mechanism may well be that of acids in
lemon or vinegar affecting the pH of the intestinal contents and inhibiting the activity
of histamine metabolising enzymes.

Amines such as histamine, cadaverine, putrescine and tyramine are found in meat and
meat products, cheeses and other fermented foods such as sauerkraut, beverages such
as wine and beer, and fruits and vegetables (Shalaby 1996), and in Chinese foods such
as tamari and soy sauce (Chin et al 1989). Therefore, it would seem that dietary items
other than fish could theoretically exacerbate HFP if consumed at the same time as
spoiled fish. A number of foodborne substances are DAO inhibitors, including
anserine, carnosine, agmatine, thiamine, cadaverine and tyramine (Taylor 1986). In
addition, the increasing popularity of mixed fish dishes such as seafood marinara may
promote HFP by providing histamine potentiators from different fish species eaten in
the same meal.

3.4.3 Influence of medication

Some drugs can inhibit histamine-metabolising enzymes and potentiate histamine
activity when taken in conjunction with food containing high concentrations of
histamine (Taylor 1986; Chin et al 1989).

50
HMT is inhibited by analogues of methylmethionine, such as adenosyl-homocysteine,
antimalarial drugs (quinacrine, chloroquin and amodiaquin) and numerous agonists
and antagonists of histamine receptors. Some antihistaminic drugs and
aminoguanidine are inhibitors of DAO. Several compounds once classified as specific
MAO inhibitors are known to inhibit DAO as well. These substances include
isoniazid. (References cited by Taylor 1986.) On the other hand, some individuals
taking antihistamines for other reasons may be protected from histamine poisoning to
some extent (Taylor et al 1989).

Isoniazid, an anti-tuberculosis medication, has been incriminated most often as an
exacerbating factor in HFP (Stratton et al 1991). Scientists in Sri Lanka have reported
several incidents of histamine poisoning among patients with tuberculosis who
consumed tuna while receiving isoniazid therapy (references cited by Taylor 1985). In
one outbreak, 21 patients were affected (Uragoda and Kottegoda 1977) and, in
another, 56 (Senanayake and Vyravanathan 1981).

3.4.4 Disease states and age

Histidine metabolism in humans can be altered in certain disease states.
Histidinaemia, an inborn error of metabolism resulting from the absence of HAL, is
associated with increased excretion of histamine and its metabolites. Patients with this
disease or with altered histamine metabolism may be more susceptible to HFP.
Exogenous histamine would also exacerbate diseases such as allergies in which
elevated endogenous histamine levels play a role, and mastocytosis (Taylor 1986).
Activities of histidine- and histamine-metabolising enzymes may also be affected with
age.

3.5 Morbidity and mortality rates

HFP is usually a rather mild illness (Taylor 1986). In rare cases it has proved fatal
(Eitenmiller 1981). Arnold and Brown (1978) report that morbidity values vary from
0.07% to 100% in the literature, but say that such figures are misleading, as different
lots of canned fish may differ greatly in their histamine content. The distribution of
histamine in individual fish is also highly variable, as is individual susceptibility to
histamine poisoning. Nevertheless, in group outbreaks the attack rate may approach
100%. When an outbreak involves raw fish, the attack rate is sometimes substantially
below 100% (Taylor 1986; Wu et al 1997).

The lack of any visual sign of spoilage in scombrotoxic fish probably accounts for the
high morbidity rate. With food allergies, it would be unusual for more than one person
in a group to experience symptoms caused by a specific food, which assists in the
differential diagnosis. Allergic reactions to some of the fish commonly incriminated in
HFP outbreaks, such as tuna and mahi-mahi, are quite rare (Taylor 1986).

51
4. EXPOSURE ASSESSMENT

4.1 Definition of exposure assessment

Buchanan et al (1998) defines exposure assessment as the probability of consumption
and the amount of the biological agent consumed. Topics that may be considered
under the heading of exposure assessment include:

levels of the hazard in food at different stages in its production;
the frequency of exposure;
the duration or extent of exposure;
exposure of sub-populations (including ethnicity, age, geographic location,
consumption patterns).

In discussing exposure assessment in relation to HFP, we will examine, in a non-
quantitative way, factors affecting the probability of HFP occurring, histamine levels
in fish products in Australia, amounts of fish consumed and at-risk population groups,
and future exposure trends.

4.2 Factors affecting the probability of histamine fish poisoning occurring

4.2.1 Post-catching contamination

There is uncertainty in the literature regarding the main source of HDB, which have
been isolated from the skin, gills, intestines and muscle tissues of spoiling fish (Lerke
et al 1978). Kimata (1961) and Yoshinaga and Frank (1982) estimated that histamine
formers occupied about 1% of the regular surface microflora of fresh fish and, with
extended storage at elevated temperatures, the bacteria invade the muscle and convert
free histidine to histamine. However, in their study of HDB contaminating fish
purchased in retail markets in Spain, Lpez-Sabater et al (1996) found that
Plesiomonas shigelloides was the sole HD isolate frequently associated with the
marine environment.

Most scientists believe that post-harvesting contamination is the most important
source of histamine formers. Taylor and Speckhard (1983) devised a method for the
recovery of HDB from frozen skipjack tuna (Katsuwonus pelamis) obtained from a
major tuna packer in the United States. Gills, intestines and muscles were sampled.
HDB (M. morganii and C. freundii) were isolated from only 3/10 fish, in each case
from the gills only. The evidence suggested that Enterobacteriaceae are not part of the
normal microflora of tuna and the isolation of these bacteria from the gills, which are
usually used as 'hand-holds' in handling, was attributed to post-harvesting
contamination. Taylor et al (1989) went further to say that most of the histamine
formers found in fish are common enteric bacteria of humans and animals. However,
the status of C. perfringens and N-group bacteria that grow at refrigeration
temperatures cannot be confirmed, since early recovery procedures used did not cater
for their detection. Scoging (1991, 1998) agreed with Taylor (1986) that outbreaks of
HFP may result from fish becoming contaminated after harvesting.

52
In the United States, most outbreaks of HFP are associated with improperly handled
fish from private catches (Lange 1988). HFP has also been linked to restaurant-
prepared meals, as well as to canned tuna, both of which involve commercial fishing
operations (Lange 1988). Post-catching contamination with HDB may occur at several
levels aboard the fishing vessel, at the processing plant, in the distribution system
(fresh and frozen fish), and at the level of the user (Taylor 1986). Contamination
during handling of canned tuna for the preparation of a tuna salad in a restaurant was
implicated in one German outbreak (Yamani et al 1981). Taylor (1986) suggests that
restaurant contamination could be particularly important with raw tuna.

To understand the sources of contamination of fish with HDB, Subburaj et al (1984)
investigated the fish market environment of Mangalore, India, for counts of these
bacteria. They cultured samples from carrying baskets, ice, the market floor, and water
for wetting fish. They also recorded total plate counts (TPC), HDB counts and
histamine levels in samples of seerfish (Scomberomorus guttatus) and mackerel
(Rastrelliger kanagurta). The results demonstrated that HDB (identified with Niven's
medium) were widely distributed in the market environment and water. Since
Morganella spp. (then Proteus spp.) are the most common bacteria associated with
HFP, the incidence of these bacteria, in particular, were examined in various market
samples (see below).

Incidence of Morganella spp. in various market samples

Sample No. analysed No. +ve for
Morganella
% of Morganella
in TPC
Water
Ice
Basket
Floor
3
3
3
4
3
1
3
4
1015
10
4080
45?

The generic composition of HDB in a mackerel sample containing 20 mg
histamine/100 g revealed Morganella (52%), Pseudomonas (21%), Plesiomonas
(12%), Providencia (8%), Flavobacteria (6%) and Aeromonas (1%). As could be
expected, there was no direct correlation between HDB count and histamine level,
confirming that the generic composition of the microflora is more important than
numbers.

If fish are stored ungutted, the gut itself may be a source of contamination, particularly
if chilling is delayed. For example, if anaerobic bacteria of the gut such as C.
perfringens (a prolific histamine producer) are allowed to proliferate at high
temperatures, they will produce enzymes that are highly active at lower temperatures
(Olley et al 1985).

4.2.2 Temperature abuse on fishing vessels

At any time between catching and consumption, exposure of certain fish to elevated
temperature can cause formation of histamine from histidine by HDB, which are
inevitably present. Quality assurance on board fishing vessels is the first link in the
quality chain that runs from the start of production to final consumption. Formation of
53
histamine can be induced by short-term temperature abuse early in the chilling step
and detected later (Andersen 1997).

The key to the reduction of histamine production is the rapid cooling of the fish after
catching (Ritchie and Mackie 1979). Initial cooling is important in reducing the rate of
histamine production, even when temperature rises at a later stage (Mitchell 1993).
Many fish species commonly implicated in outbreaks of HFP are caught in warm
water, and fish temperatures at capture frequently exceed 20
o
C. Fish may remain in
purse-seiner nets or on longlines at this elevated temperature for several hours after
capture, and fish may die before they leave the water.

Tuna have higher body temperatures than most other fish. In the western Pacific,
where sea surface temperatures are 2730
o
C, tuna often come on board with internal
temperatures of 32
o
C. Spoilage at this temperature is about 30 times faster than at
0
o
C. Because of elevated temperatures, the length of time from death until tuna are
chilled is the most critical phase of shipboard handling (Bartram 1997).

Once on board the fishing vessel, the fish may or may not be cooled, and methods of
cooling employed vary widely in their efficiency (Taylor 1986; Price 1999). American
purse seiners cool the fish in holds filled with refrigerated seawater. Once the hold is
full, the fish can be frozen. The rate of cooling depends on the size of the catch and on
the size of individual fish. The fish may be held at elevated temperatures for some
time, and are not gutted until they reach the processing plant. Variable conditions exist
from boat to boat, and this probably explains the sporadic incidence of histamine
poisoning (Taylor 1986). Any time a fish is held at >4.4
o
C (40
o
F) significantly reduces
the expected safe shelf life, but fish that have been handled particularly well on-board
the harvest vessel may be able to safely withstand somewhat more exposure to
elevated temperatures during post-harvest handling (Price 1999).

4.2.3 Inadequate chill-storage procedures

Taylor (1986) cites a number of studies on the effect of storage temperature on
histamine formation in various types of fish. While all the studies agreed that
histamine formation is negligible in fish stored at 0
o
C or below, other results were
variable. Widely varying data exist for both the lower temperature limits for safe
storage and for the optimal temperature for producing histamine. This is not
surprising, given the variability of the nature of the bacterial populations on fish.

Low-temperature storage (<10C) effectively controls the growth of most histamine-
producing bacteria, which require a warm temperature for growth. However, bacteria
that grow at refrigeration temperatures can produce smaller amounts of histamine in
fish stored at temperatures between 0 and 10C (Ritchie and Mackie 1979; Klausen
and Huss 1987b; Stratton and Taylor 1991). Regardless of the species involved,
bacteria must grow to a large enough population for significant production of
histamine to occur.

In fish subjected to elevated temperatures, even for short periods, a large population of
bacteria is soon established. During subsequent refrigeration, although bacterial
growth ceases, residual enzyme activity continues slowly and histamine levels
54
continue to increase (Klausen and Huss 1987b; Stratton and Taylor 1991; Institute of
Medicine 1991). Klausen and Huss (1987b) found that large amounts of histamine
were formed in histidine-containing broth and in mackerel at low temperatures (0
5
o
C), where no growth of M. morganii took place, following storage at higher
temperatures (1025
o
C).

Geiger (1944a) found that histamine concentration in scombroid fish increased from
0.09 mg/100 g when fresh to 95 mg/100 g when kept at room temperature (2025
o
C)
for 10 h. According to Gilbert et al (1980), fish packed in melting ice (0
o
C) remain
edible for about 12 days, by which time histamine concentrations are still only 3
4 mg/100 g. At a room temperature (1525
o
C), high concentrations of histamine are
reached rapidly, and fish become toxic, even though they may appear acceptable to the
consumer.

Ritchie and Mackie (1979) monitored the formation of histamine, putrescine,
cadaverine, spermine and spermidine in freshly caught mackerel and herring held
ungutted on ice for 28 days at 1
o
C, in an incubator at 10
o
C, or in an insulated box at
ambient temperature (25
o
C). In mackerel held at 1
o
C, histamine rose slowly from an
initial level of 0.01 mg/100 g at day zero to 5.25 mg/100 g at day 14, and quickly to
57.94 mg/100 g at day 28. Cadaverine rose from 0.01 mg/100 g at day zero to
43.08 mg/100 g at day 28, while putrescine rose from 0.05 mg/100 g to
8.92 mg/100 g. Similar trends occurred for herring. The ratios of the final
concentration of histamine:cadaverine:putrescine at day 28 for mackerel and herring
stored at 1
o
C were 6.5:4.8:1.0 and 8.3:5.9:1.0 respectively. Thus, the concentrations of
histamine and other amines, even in putrid fish, were quite low (less than
100 mg/100 g/fish) after prolonged low-temperature storage. At the higher
temperatures, the amines were produced in relatively large amounts.

In an experiment involving nine lots of sardines with undocumented post-harvest
history bought at a fish market in Morocco, Ababouch et al (1986) demonstrated the
effect of refrigeration on histamine development. Sardines stored at 8
o
C had a 12-h
longer shelf life than those held at 17
o
C. A combination of salting and refrigeration
was more effective. Fish held at 8
o
C and salted at a level of 5% or 8% had a shelf life
35 h longer than fish held at 17
o
C with no salt.

Chen and Malison (1987) reported typical scombroid symptoms after consumption of
mackerel stored on ice for 2 days and then kept at room temperature of 30
o
C for 34 h
before cooking. The incriminated fish contained only 10 mg/100 g histamine. Toxin
production apparently occurred at a rapid rate, because people who ate lunch from
12.30 to 1 pm had a much higher attack rate than those who ate 12 h earlier. Other
fish from the same catch did not cause illness after standing at room temperature for
only 1 h.

4.2.4 Inadequate freezing and thawing procedures

Ben-Gigirey et al (1998) investigated the changes in biogenic amines, and numbers of
bacteria reported to have decarboxylase activity, in albacore (white tuna) muscle
during frozen storage. Albacore specimens of high quality (either 'extra' or A category
on sensory analysis) were analysed for their biogenic amine and bacterial contents
55
after 1, 3, 6 and 9 months of frozen storage at 18
o
C or 25
o
C. Thirty-seven intact
specimens of albacore were purchased in October 1995 at the docks of Burela (north-
western Spain) and transported on ice and under refrigeration (4
o
C) to the laboratory.
The weight range was 1.23.3 kg. Five samples were kept on ice 'until samples could
be taken for microbiological and biochemical analysis'. The remaining 32 fish were
glazed in cold water, frozen in a freezing tunnel at 40
o
C, and divided into two groups
of 16 fish each that were stored separately at 18
o
C and 25
o
C.

The scientists optimised a HPLC method involving a linear elution gradient for the
identification and determination of putrescine, cadaverine, histamine, spermine and
spermidine. The initial average contents of all the amines, except for spermidine, were
<0.8 mg/100 g. Putrescine decreased during storage after 6 months at either
temperature, but increased to average concentrations of 5.9 mg/100 g (815% of the
initial level) and 6.8 mg/100 g (942% of the initial level) in the white muscle of
albacore after 9 months of frozen storage at 18
o
C or 25
o
C respectively. Histamine
decreased after 3 months storage, but at 18
o
C it increased to a final concentration of
0.48 mg/100 g (103% of the initial level) after 9 months of storage. This rise did not
occur in samples stored at 25
o
C.

Cadaverine contents tripled or doubled after 3 months of storage at 1
o
C or 25
o
C
respectively. Thereafter, cadaverine levels began to decrease. After 9 months' storage,
they were higher at 25
o
C (0.26 mg/100 g, 86% of the initial level) than at 18
o
C
(0.17 mg/100 g, 56% of the initial level). Spermine contents increased as frozen
storage progressed, and reached concentrations of 0.95 mg/100 g (120% of the initial
level) and 1.0 mg/100 g (129% of the initial level) after 9 months at 18
o
C and 25
o
C
respectively. By contrast, spermidine contents persisted in the 1112.8 mg/100 g
initial range during the first 6 months of frozen storage, and after 9 months the final
concentrations of this amine were significantly lower.

No pathogens responsible for foodborne disease were isolated. Aerobic bacteria that
grow at ambient temperatures survived frozen storage at 18
o
C (39%) and 25
o
C
(92%) for 9 months. The survival rate at 25
o
C of bacteria that grow at refrigeration
temperatures was 4.6% after 9 months.

The results suggest that good handling practices are mandatory during the thawing of
fish before canning, since the absence of certain bacteria potentially involved in
spoilage due to amines is not guaranteed, even after 9 months' storage at 25
o
C, and
the levels of some biogenic amines may increase.

4.2.5 Temperature abuse in the preparation of dried and/or smoked products

Toxic levels of histamine have been found in dried and/or smoked products of
mackerel, horse mackerel and sardines (Taylor 1983), as well as in the fishmeal made
from these fish. Exposure of raw fish to high ambient temperatures accelerates this
reaction. Histamine content increases to a maximum, then decreases with prolonged
drying time. The drying of sardines previously brined in 515% sodium chloride for
2 h causes pronounced increases in histamine (Pan 1988).

56
Trinidad et al (1986) reported on histamine production in Spanish mackerel smoked
for 8 h at 45
o
C and stored at refrigeration temperature (05
o
C) as 3-mm-thick slices
packed in flexible plastic films, under vacuum or not. Histamine increased in both
kinds of packs (see table below), but not to a significant extent because the fish used
for smoking were very fresh, kept chilled, and contamination during handling and
processing was kept to a minimum. Vacuum-packed samples had lower microbial
counts at the middle of storage, but at the end (day 30), the difference between the two
was negligible.

Changes in histamine content (mg/100 g) of sliced smoked mackerel
after storage at 05
o
C

Storage day Vacuum-packed Non-vacuum-packed
0
14
30
1.12
2.18
3.19
1.12
2.98
4.20

Van Spreekens (1987) reported high levels of histamine in vacuum-packed, lightly
salted herring fillets stored at refrigeration temperatures, particularly after low-level
contamination with histamine-producing photobacteria. The salt concentration of the
product was a vital factor in their growth (0.54%).

Van Spreekens (1987) attributed histamine production in salted or hot-smoked
mackerel to Photobacterium spp., which grow at refrigeration temperatures. The fish
were exposed to temperature abuse as a result of delayed cooling on board ship and/or
during processing. Van Spreekens (1987) maintains that, because photobacteria are
thermolabile and have special requirements for growth, they have often been
overlooked as HDB and causative agents in HFP.

Hot smoking 'practically sterilises' the product and denatures enzymes, thus imparting
some degree of preservation, but does not destroy histamine already formed (Poulter
1988). Bremer et al (1998) carried out thermal death trials using a H. alvei strain
isolated from a portion of hot-smoked kahawai with a histamine level of
166 mg/100 g. Results of the trials, carried out in 0.1% peptone suspension and in
kahawai at 54, 55, 56, 57 and 58C, indicate that hot smoking has the potential to
eliminate H. alvei from seafood products. At the same time, histamine is very resistant
to heat and, once present in a fish, is not easily destroyed by smoking, cooking or
canning.

Fletcher et al (1998) reported the results of a retail survey of the levels of histamine in
hot-smoked fish products available in New Zealand, where most cases of HFP are
associated with smoked fish. They purchased 107 samples from Auckland retail
markets between July 1995 and March 1996 and determined their histamine and
bacterial levels. Eight samples (from 5/9 retail markets) had histamine levels that
exceeded 50 mg/kg. In two samples, histamine levels exceeded 200 mg/kg (346 and
682 mg/kg). Within or between fish species there were no consistent relationships
between levels of histamine in the samples and either the total aerobic plate counts or
the numbers of histamine-producing bacteria. Some of the samples with elevated
levels of histamine had relatively low microbial counts. For example, sample 136 had
the highest histamine level of any of the retail products, yet its microbial counts were
57
all less than 6 x 10
3
CFU/g. The evidence showed that histamine had been formed
prior to smoking, and that histamine-producing bacteria were eliminated during
smoking.

4.2.6 Poor canning procedures

The rate of histamine formation in fish prior to canning is a function of:

temperature during storage and preparatory operations;
duration of exposure at critical temperatures;
contamination levels of HDB; and
levels of free histidine in the fish (Warne 1985).

Incidents of HFP caused by high levels of histamine in commercially canned
scombroid products have occurred all over the world (Taylor 1983). Improvements in
handling and processing associated with the establishment of HACCP procedures are
becoming widespread. These offer the advantage of in-process control, while avoiding
the need for large-scale terminal analyses of finished product, which is time
consuming and beyond the capabilities of many small canneries (Warne 1985).

4.2.7 Low-quality fermented products

Fermented fish containing high levels of histamine could conceivably present a
hazard. In Finland, sugarsalted herring in barrels is more likely to contain high levels
of histamine than canned herring, but has not been implicated as causing illness. More
research is needed to define suitable limits for histamine in fermented products
(Taylor 1985). Fermented fish products are usually consumed in small amounts, so
higher concentrations of histamine could possibly be tolerated in these products.

4.2.8 Temperature abuse of raw tuna for the sashimi market

The principal seafood safety hazards in the lucrative raw tuna trade are decomposition
and histamine formation. Both are linked to temperature abuse. Raw tuna buyers are
presented with so much quality variation that each fish is inspected and graded prior
to purchasing. The primary purpose of this evaluation is economic to direct each
tuna to the most profitable market niche. When decomposition or high histamine
levels occur, they usually involve the lowest grade (Grade 4 or D) of raw tuna. The
highest quality raw tuna is sold in sushi bars. Grading fulfils the objectives of
HACCP, because raw tuna core temperatures are checked and fish are
organoleptically examined at critical control points (Bartram 1997).

58
4.3 Histamine levels in fish products in Australia
4.3.1 Survey of histamine in canned tuna
In the absence of information concerning the prevalence of histamine in canned tuna
consumed in Australia, Warne et al (1987) screened cans of tuna purchased from
several Melbourne retail markets. Of 104 cans surveyed, 45 were produced in
Australia and the remainder came from Japan, Malaysia, the Philippines and Thailand.
Product from each of the cans was screened for histamine at <5 mg/100 g, 5
10 mg/100 g and >10 mg/100 g using TLC based on the method used at the Torry
Research Station in the United Kingdom.

Most cans (101/104) contained <5 mg/100 g, and therefore complied with the most
stringent of current international standards. In 3/104 cans, histamine levels were in the
510 mg/100 g range, which is still acceptable. During canning, the HD enzyme is
denatured at retorting temperatures, while bacteria responsible for its production are
killed. The presence of >5 mg/100 g in 3/104 samples suggests that fish were
mishandled at some stage in the sequence of operations leading up to retorting.

4.3.2 Survey of biogenic amines in fish products in the ACT

Another survey was published as an information paper on the Internet. The Australian
Capital Territory Government published the results of a survey of biogenic amines in
fish and fish products from April to June 1997 (Rigg 1997). Objectives were to
determine:

the compliance of fish and fish products (including fresh, canned and dried) for
histamine to the Australian Food Standards Code (ANZFA 1998); and
the levels of two other biogenic amines, putrescine and cadaverine, which are also
of concern in fish products.

The paper was poorly presented and did not have a 'Materials and methods' section.
Analytical methods were not given, and 'detectable amounts' were not specified. 'Fish'
were not adequately defined and included molluscs, prawns and seafood marinara.
The paper stated that 58/64 of samples (91%) did not have detectable levels of
histamine, 5/64 (8%) contained levels below the Food Standards Code level of
100 mg/kg and 1/64 (2%) failed to comply with the standard. The latter sample, of
dried fish imported from Asia, contained 653 mg/kg histamine. The paper went on to
say that 3.4% (1/29) of fresh 'fish' compared to 14.3% (5/35) of processed fish
samples were found to contain histamine. These samples were one of fresh chunk
tuna, one of tinned fish and four of dried fish. Histamine was not detected in any of
the 20 samples of crustaceans and molluscs surveyed. There was a significant
difference in histamine levels between fresh (3.3%) and processed (14.7%) fish
products.

Putrescine was found in 20/64 samples (31%) and cadaverine in 28/64 (44%). For
putrescine, 10/29 of fresh samples (34%) and 10/35 of processed samples (29%) had
detectable amounts. There were five samples that exceeded 100 mg/kg, which
included two of green prawns, one of scallops and two of dried fish. For cadaverine,
59
13/35 (37%) of fresh samples and 15/35 (43%) of processed samples had detectable
amounts. Three contained >100 mg/kg two of dried fish and one of scallops.

4.3.3 Monitoring by the Australian Government Analytical Laboratories

The Australian Government Analytical Laboratories (AGAL), Melbourne, monitor
canned and raw tuna and other fish imports for the Australian Quarantine and
Inspection Service (AQIS). Random samples are analysed on a regular basis and
included fish from various Asian countries and the United States. The detection
method used is capillary electrophoresis (Trenerry et al 1998), which has proved more
satisfactory than the AOAC fluorometric method (AOAC 1995c). Only a small
percentage of samples exceed the Australian Food Standards Code (ANZFA 1998)
level of 100 mg/kg, and in these few samples concentrations are rarely much greater
than 100 mg/kg (Philip Marshall, AGAL, pers. comm. 1999).

4.3.4 Monitoring by industry

Information on industry monitoring for histamine levels in fish and fish products was
sought from Port Lincoln Tuna Processors Pty Ltd, South Australia, the only
remaining tuna cannery in Australia. Skipjack tuna is canned at Port Lincoln. All
batches entering the cannery (mainly from Australia, but some from overseas) are
monitored, with five fish from each batch being tested. The factory also monitors
histamine in random samples of its canned product. The ALERT
TM
ELISA test kit is
used (sensitivity 50 ppm). In addition, every 6 months, samples are sent to AGAL in
Melbourne for analysis by capillary electrophoresis (sensitivity 10 ppm), to ensure
standardisation of results. The two methods of analysis show good agreement, and
histamine levels routinely do not exceed the Australian Food Standards Code
maximum level of 10 mg/100 g (Lea Traeger, pers. comm. 1999).

4.4 Amounts and types of fish consumed, and at-risk population groups
All population groups are susceptible to HFP, but regional differences undoubtedly
exist in the amounts and types of fish consumed and the way they are processed and
stored. The disease still occurs frequently, even in developed countries such as the
United States, Japan and the United Kingdom (Taylor 1986).
Figures were not found for consumption of various fish species in different countries.
Even if they were found, their value would be limited in assessing possible exposure
to HFP, because details of handling and storage conditions would not be known.
Although fish consumption in Australia is probably increasing, as people are
becoming more health conscious, it is not high by world standards. In 1995, ANZFA
conducted an Australian National Nutrition Survey based on a 24-h recall survey
method for 13 858 people aged 2 years and over. Total mean intake of all species of
fish for all respondents (consumers and non-consumers of fish) was 12.2 g/day, and
for marine fish the figure was 10.2 g/day. This means that the average Australian
consumes less than 4.5 kg/year fish in his/her diet and less than 4 kg/year of marine
fish. Although tuna probably represents a fairly high proportion of the marine fish
consumed, because of its commercial importance, the chance of an Australian
contracting HFP is still very low.
60
HFP is probably still common in developing countries where fish preserved by
traditional methods is an important part of the diet. Such diets remain extremely
popular and are a major source of inexpensive dietary protein. Their popularity is
largely due to desirable flavours produced by chemical changes that occur during the
smoking and drying processes. Traditional African products are rarely salted, but
some may be sun-dried before smoking starts. In the course of sun and air drying,
bacteria can multiply in the moist interior of the fish, which has high water activity,
causing the inside to spoil while the outside appears satisfactory. The problem would
be expected to be greater in tropical countries where higher ambient temperatures
promote the growth of prolific HDBs (Fig. 3).
In tropical countries, fish are cold smoked at 3040oC, depending on species, to
prevent coagulation of protein, which occurs with cooking. This process is designed to
give a desired flavour to the product rather than a significant degree of preservation
(Poulter 1988). It will not destroy HDB or deactivate HD. Even if hot smoking
follows cold smoking, histamine concentrations may have already become high
enough to become hazardous.
Products cured in the traditional way have become less popular in most developed
countries. However, in some non-tropical countries, such as the United Kingdom, fish
are cold smoked at low temperatures (<30
o
C depending on species, Anon. 1988a).
The modern, mildly smoked and less extensively dried products are less stable than
the traditional ones, and this trend has been made possible by chilling and freezing.
Neither cold- nor hot-smoked fish will keep for long periods unless supplementary
techniques, such as drying, freezing or chilling, are used (Poulter 1988).
The consumption of raw fish, mainly by the Japanese, probably presents less risk of
HFP because only the highest quality fish are directed to this trade. Also, as the
product is a delicacy, smaller quantities of fish would be likely to be consumed per
meal.
4.5 Future exposure trends
In the past 10 years or so, authorities and producers around the world have become
more aware of the need for quality assurance in relation to food (Barker and
McKenzie 1997). As a result, standards for seafood quality are improving and
exposure to spoiled seafood and thus HFP is likely to become less prevalent.

Developing countries have increased their net income from fish and fishery products
from about US$3 billion to some US$18 billion (FAO 1999) over the past 10 years.
By 1997, developing countries had equalled the production of the developed world,
contributing almost 50% of global fishery exports (Karnicki 1997). Great progress has
been made in the quality of fish products at the same time as the huge expansion of
international trade. This is the result of the introduction of international standards in
food hygiene and the application of risk analysis and HACCP principles, as part of a
common, global approach for maximising the quality and safety of all food products
(FAO 1999). Thus, HFP is now much less likely to occur as a result of commercial
61
canning or smoking as HACCP quality assurance measures are increasingly being
adopted.

In Australia, the Australian Quarantine and Inspection Service (AQIS) has introduced
the Hazard Analysis and Critical Control Point (HACCP) and International Standards
Organization 9000 series of standards (ISO 9000) quality management inspection
systems. These initiatives, together with those of other government and industry
agencies, have encouraged the Australian export seafood industry and other sectors of
the processed food industry to implement quality management systems that address
issues such as good handling practices and temperature control. AQIS, as part of the
Commonwealth Department of Agriculture, Fisheries and Forestry, will endeavour to
ensure that the application of HACCP-based inspection systems in Australia remains
consistent with overseas regulatory authorities (Aitken 1997).
Predictive microbiology is an emerging area of food microbiology in which microbial
responses to environmental factors are measured under defined and controlled
conditions. The potential applications of predictive modelling in HACCP systems are
numerous (Ross and McMeekin 1995). Although predictive modelling cannot be
applied to histamine production directly, for reasons discussed in section 3.3.5.2, it
has an important future role in the potential to control microbiological spoilage of fish
and thus will assist indirectly in the control of HFP.
5. RISK CHARACTERISATION
5.1 Definition of risk characterisation
Kindred (1996) defines risk characterisation as The description of the nature and
often the magnitude of human risk, including attendant uncertainty. According to
Buchanan et al (1998), this involves integration of the doseresponse and exposure
assessments into a risk statement, which includes estimation of the likelihood and
magnitude of disease under various conditions of exposure and description of
uncertainties. The risk characterisation should result in a quantitative or qualitative
estimate of the potential for adverse effects of the particular biological hazard on the
population.
In this section, an attempt will be made to summarise the nature and magnitude of the
risk of HFP, health and other impacts of the disease, and uncertainties and problem
areas in understanding and combating the disease. Although much is known about
HFP, there are still many gaps in our knowledge. Sufficient data are not available at
this stage for an adequate risk assessment. Appropriate strategies of risk management
and risk communication can be put in place only after further data are generated and
analysed.
62
5.2 Nature and magnitude of risk
5.2.1 Impact on human health
HFP does not have a major impact on human health, at least in Australia. It is a mild
but unpleasant disease, likely to cause discomfort for only a few hours. The morbidity
rate may approach 100% following the consumption of toxic fish, but mortality is
extremely rare. The disease may be more serious in people taking certain medicines or
with pre-existing pulmonary, cardiac or renal problems.
The reports of HFP in Australia and New Zealand in the scientific literature (Smart
1992; Brown 1993; Foo 1975a; Mitchell 1993) are not sufficient to indicate the
frequency of occurrence of HFP in these countries. To rely on these reports for
information would suggest that the disease is rare. Because HFP is generally mild and
of short duration, and responsive to antihistamine therapy, most outbreaks are unlikely
to be reported, even in the daily press.
However, HFP is important from the food safety aspect. It is possible that products,
particularly imports, will escape the random monitoring safety net from time to time.
Consumers are becoming more demanding, and do not expect food to make them ill.
Litigation following food poisoning incidents is becoming more common and
producers, distributors and restaurants will increasingly be held liable for the quality
of the products they handle and sell.
5.2.2 Impact on fishing industries
In 199697 Australia produced fishery products worth $1760 million, with exports
worth $1290 million. Total finfish production was about 127 kt worth $454 million
(ABS 1999).
If a major outbreak of HFP were to occur in Australia, as they have in Japan and the
United States in the past, resulting media attention would affect fish consumption and
have a negative impact on the marketing of seafood. An outbreak in another country
caused by our exports would seriously affect trade. Although such events are
becoming increasingly less likely because of the widespread adoption of HACCP
analysis and quality assurance, they will still occur occasionally owing to incidents
such as those caused by equipment failure, human error or negligence.
5.3 Uncertainties and problem areas in risk characterisation
5.3.1 Defining histamine fish poisoning and elucidating its pathogenesis
The mechanism of toxicity of HFP is still not clearly understood, which is
unsatisfactory. More research is needed to determine the threshold of toxic dose for
histamine and the role that potentiators or other toxins may play in causing the
disease. It is particularly important that HFP be clearly differentiated from syndromes
caused by endogenous toxins such as ciguatoxins that may be present in finfish from
time to time.
63
As suggested by Mitchell (1993), there may be potential in fractionation of the
chemical constituents of toxic fish and assessment of the toxicity of different single
and combined fractions. In addition, it is the view of the authors that little attention
has been given to the pKa values of the carboxyl, imidazole and amine groups of the
compounds discussed in this review, although pKa greatly influences biological
properties. A list of pKa values can be found in any handbook of data for biochemical
research (e.g. Dawson et al 1969), and is the pH at which half of the ionisable group is
in the dissociated and half is in the unionised form. It is significant that the pKa of the
imidazole group is in the normal physiological range. Similarly, in separating these
compounds found in spoiling fish chromatographically, little consideration has been
given to the pH of the solvent or to the stabilising effects of solvent counter-ions.
Future regulatory action should take into account the impact of potentiators, and the
identification of potentiators in various foods may be essential (Hui and Taylor 1985).
When analysing suspect fish for histamine, there may be advantage in simultaneous
detection of other common putrefactive amines, such as cadaverine and putrescine
(Taylor and Sumner 1986).
The safety of urocanic acid has been investigated only in relation to its use in cosmetic
products and sunscreens (Cosmetic Ingredient Review Expert Panel 1995). Most
animal experiments have involved topical administration and the toxicity profile is
incomplete. Extensive animal data indicated that the substance has
immunosuppressant properties, but clinical data were inconclusive. The Cosmetic
Ingredient Review Expert Panel reported that it cannot be concluded that urocanic
acid is safe in cosmetic formulations. The possible role of urocanic acid in HFP, as a
potential mast cell degranulator, may be a suitable research project, beginning with
oral dosing studies in laboratory animals.
When the pathogenesis of the disease is understood, clinical HFP will be able to be
defined more accurately and making a differential diagnosis will be easier. There is a
need to assess the actual incidence of fish allergy and to determine what percentage of
cases diagnosed as fish allergy represent misdiagnosis and misclassification of HFP.
The latter is differentiated from allergy by the occurrence of clusters of affected
people rather than single cases, and by the application of skin tests using non-toxic
fish extracts.
5.3.2 Investigating and managing post-harvesting contamination
More research still needs to be done on post-harvesting contamination in order to
improve quality control procedures. Identification of points at which temperature
abuse and/or bacterial contamination occur would include the gathering of
information, where this has not been done already, on current fish harvesting,
transporting, storage, processing and retailing practices for the species identified as
those most likely to cause HFP. For example, temperature abuse may occur on
recreational boats that lack adequate refrigeration; and Gellert et al (1992) suggested
that rinsing fish in seawater during handling and cleaning could promote
contamination with microorganisms capable of metabolising histidine to histamine.
64
Regardless of the origin of the spoilage bacteria, histamine serves as an indicator of
microbial spoilage in histidine-containing fish, and many countries including
Australia have defined maximum histamine levels for fish products. Accumulation of
extensive data on levels of histamine in retail fish and fish products in Australia
would produce a better understanding of the likely incidence of HFP and provide a
reference point to measure future food hygiene standards. This could be carried out
either in special surveys or the work could be included in routine sampling programs
for other contaminants. Given the variety of species that may potentially contain high
levels of histamine, perhaps all finfish should be monitored. A rapid and cheap assay
for detecting histamine in fish would be of value if made available to the public and,
in particular, to recreational fishers. The current ELISA tests are rapid, but not cheap
(Lea Traeger, pers. comm. 1999).
Histamine analysis as a means of monitoring spoilage is associated with a number of
problems. It is time consuming and/or expensive, and samples need to be taken from
multiple sites to compensate for variations in histamine content throughout the
decomposed fish. Perhaps more important is the lack of standardisation of histamine
detection methods employed around the world, and the multitude of tests available.
There is a need for global standardisation of histamine detection methods, and
laboratory accreditation and proficiency testing. For all these reasons, together with
the fact that monitoring fish histamine levels may not always ensure protection from
HFP, a method other than direct measurement of histamine may be preferable for
quantitative measurement of quality deterioration. Baronowski (1985) suggested that
urocanic acid may be a useful alternative to histamine as a spoilage index in
scombroid and other fish that are rich in endogenous histidine. This idea should be
investigated, especially in the light of new knowledge that urocanic acid is a mast cell
degranulator (Wille et al 1999).
A study of the correlation between the amine content and bacterial counts needs to be
done. Ideally, each amine would be correlated with its respective amine-producing
bacteria. The detection of histamine, cadaverine and putrescine can be achieved
satisfactorily by TLC or HPLC. As for the development of a medium for detection and
enumeration of the amine-producing bacteria, only HDB have received considerable
attention. Rapid tests for lysine decarboxylase activity (Brooker et al 1973) and
ornithine decarboxylase activity (Fay and Barry 1972) could give results within 4 h of
incubation, but a solid medium that would allow direct enumeration of these activities
is not available (Leung 1987).
Government regulatory agencies require the removal of contaminated fish from the
marketplace when human illness occurs and the fish is demonstrated to contain
unusually high levels of histamine. For this reason, reporting of suspected cases of
HFP to local food authorities can lead to removal of contaminated fish from the
marketplace and prevention of additional cases.
Mechanisms need to be in place to allow efficient and complete traceback of
incriminated fish to point of origin, in order to rectify problems leading to spoilage. At
present in Australia, tracing fish from commercial outlets is difficult, because of a
lack of a ticketing system, especially in the middle (wholesaler) stages of distribution
65
(Rawlin and Herfort 1999). This is a problem area that could be improved
considerably, given the necessary resources and cooperation between stakeholders.
Quality control programs operated by the fish processing industry can lead to the
removal of contaminated fish from the marketplace. For example, such programs in
the tuna canning industry have largely eliminated histamine poisoning as a concern
with canned tuna (Taylor et al 1989; Lea Traeger, pers. comm. 1999).
66
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Appendix 1: Acknowledgments
Queensland Department of Primary Industries/National Seafood Centre
(Brisbane)
Stephen Thrower
Sue Poole
Australia and New Zealand Food Authority
Peter Abbott
Janis Baines
Scott Crerar
Australian Government Analytical Laboratories (Melbourne)
Philip Marshall
Agriculture, Fisheries and Forestry Australia
Albert Caton
Terry Nicholls
Mike Nunn
Port Lincoln Tuna Processors Pty Ltd
Lindsay Guillot
Lea Traeger
University of Tasmania
Christian Garland
John Bowman
Tom McMeekin
Tom Ross
Elisa Systems
Rob Sherlock

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