3. QUALITY ASPECTS ASSOCIATED WITH SEAFOOD

In this chapter only the quality aspects related to safety and spoilage of seafood is discussed. The various disease agents which have been associated with consumption of seafood are listed and a few characteristics relevant to the evaluation of the hazards and risks related to their presence on fish and fish products are presented. The processes leading to spoilage and control options for disease agents as well as spoilage processes are briefly outlined.

3.1. PATHOGENIC BACTERIA

Seafood borne pathogenic bacteria may conveniently be divided into two groups as shown in Table 3.1.

1) Other vibrios are: V. vulnificus, V. hollisae, V. furnsii, V. mimicus, V. fluvialis.
2) For verotoxin-producing strain 0157:H7

3.1.1. Indigenous bacteria (Group 1)

The bacteria belonging to the group 1 are common and widely distributed in the aquatic environments in various parts of the world. The water temperature is naturally having a selective effect. Thus the more psychrotrophic organisms (C. botulinum and Listeria) are common in Arctic and colder climates, while the more mesophilic types (V. cholerae, V. parahaemolyticus) are representing part of the natural flora on fish from coastal and estuarine environments of temperate or warm tropical zones.

It should be emphasized, however, that all the genera of pathogenic bacteria mentioned above contain non-pathogenic environmental strains. For some organisms it is possible to correlate between certain characteristics and pathogenicity (e.g. the Kanagawa-test for V. parahaemo-lyticus) while in others (e.g. Aeromonas sp.) there are no known methods available.

While it is true that all fish and fish products which have not been subject to bactericidal processing, may be contaminated with one or more of these pathogens, the level of contamination is normally quite low, and it is unlikely that the numbers naturally present in uncooked seafood are sufficient to cause disease. An exception is the cases when pathogens are concentrated due to filtration (molluscs). On the other hand high levels of group 1 bacteria may be found on fish products as a result of growth. This situation is constituting a serious hazard with a high risk of causing illness. Growth (and possible toxin production) must therefore be prevented. Some of the growth requirements of group 1 organisms are listed in Table 3.2. Some essential characteristics concerning each of the listed organisms are discussed below.

Clostridium botulinum

C. botulinum is widely distributed in soil, aquatic sediments and fish (Huss 1980, Huss and Pedersen 1979) as shown in Figure 3.1.

Human botulism is a serious but relatively rare disease. The disease is an intoxication caused by a toxin pre-formed in the food. Symptoms may include nausea and vomiting followed by a number of neurological signs and symptoms: visual impairment (blurred or double vision), loss of normal mouth and throat functions, weakness or total paralysis, respiratory failure, which is usually the cause of death.

Epidemiology and risk assessment

Examination of 165 outbreaks of botulism caused by fish products showed that the lightly preserved products (smoked, fermented) represented by far the most dangerous group as shown in Table 3.3.

1) Shultz et al. (1984).
2) Delmore and Crisley (1979).
3) Farber and Peterkin (1991).
4) Nolan et al. (1992)
5) London et al. (1992)
6) Conner et al. (1989)
7) Miller and Koburger (1986)

In contrast it should be noted that fresh and frozen fish has never been shown to cause human botulism. This is probably due to the fact that fresh fish normally spoil before becoming toxic. The ultimate safeguard is very low heat stability of botulinum toxin (Huss 1981, Hauschild 1989), which means that normal household cooking will destroy any preformed toxin. Thus the risk is clearly associated with foods that do not require cooking immediately before consumption.

Disease control

Botulism may be prevented by inactivation of the bacterial spores in heat-sterilized, canned products or by inhibiting growth in all other products. C. botulinum is classified into toxin types from A–G, and the types pathogenic to man can conveniently be divided into 2 groups:

1. The proteolytic types A and B, which are also heat-resistant, mesophilic and NaCl-tolerant.

2. The non-proteolytic types E, B and F, which are heat-sensitive, psychrotrophic and NaCl-sensitive. It is primarily the non-proteolytic types which are found on fish and fish products.

Canning processes have generally been designed to destroy a large number of the heat-resistant C. botulinum types. Thus the “botulinum cook” has been defined as equivalent to 3 min. at 121°C. This value is also called the F_o value or the “process value”. The F, value required for canned fish product is equivalent to 12-decimal reductions of Clostridium botulinum spores. Using the highest known D-values (0.25 min at 121°C) the F_o is therefore equal to 12 x 0.25 = 3. This is the so-called 12 D-concept designed to reduce the bacterial load of one billion spores in each of 1000 cans to one spore in a thousand cans.

Figure 3.1. Incidence (%) of C.botulinum in fish. Letters A-F indicate the presence of C.botulinum type A-F. For references to the various surveys see Huss (1980).

In contrast, the D-value for the non-proteolytic group is much lower. Based on data presented by Angelotti (1970) a wet heat process of 82,2°C for 30 min. should destroy approx. 10⁷ spores. Commercial heat pasteurization (sous-vide products, hot smoking) may therefore not be sufficient to kill all spores and safety of these products must be based on full control of growth - and toxin production.

Some of the most important limitations for growth for C. botulinum are listed in Table 3.2. Although it is shown that the non-proteolytic strains can grow in up to 5% NaCl this is only the case under optimal conditions. In fish products stored at low temperature (10°C), 3% NaCl in the water phase is sufficient to inhibit growth of type E for at least 30 days (Cann and Taylor 1979). Table 3.4 summarize the most important safety aspects of the various types of fish products.

Vibrio sp.

Most vibrios are of marine origin and they require Na⁺ for growth. The genus contains a number of species which are pathogenic to man as listed in Table 3.1. V. cholerae occurs in two serotypes, the 01 and the non-01, and the 01 serotype occurs in two biovars: the classic and the El tor. The classical biovar, serovar 01 is today restricted to parts of Asia (Bangladesh), and most cholera is caused by the E1 tor biovar. The pathogenic species are mostly mesophilic, i.e. generally occurring (ubiquitous) in tropical waters and in highest numbers in temperate waters during late summer or early fall.

The diseases associated with Vibrio sp. are characterized by gastro-enteritic symptoms varying from mild diarrhea to the classical cholera, with profuse watery diarrhea. One exception is infections with V. vulnificus, which are primarily characterized by septicaemias.

The mechanisms of pathogenicity for the vibrios is not entirely clear. Most vibrios produce powerful enterotoxins and as little as 5μg cholera toxin (CT) administered orally caused diarrhea in human volunteers (Varnam and Evans 1991). A number of other toxins are produced by V. cholerae, including a hemolysin, a toxin similar to tetrodotoxin and one similar to shiga-toxin. Pathogenic strains of V. parahaemolyticus are known to produce a thermostable direct hemolysin (Vp-TDH), which are responsible for the Kanagawa-reaction, but it is now documented that also Kanagawa-negative V. parahaemolyticus are able to produce disease (Varman and Evans 1991).

The named pathogenic Vibrio sp. are not always pathogenic. The majority of environmental strains lack the necessary colonization factors for adherence and penetration, appropriate toxins or other virulence determinants necessary to cause disease.

In recent years it has been demonstrated that vibrios are able to respond to adverse environmental conditions by entering a viable, but non-culturable phase (Colwell 1986). When the bacteria are exposed to adverse conditions of salinity, temperature or nutrient deprivation, they can be reversibly injured and they cannot be detected by standard bacteriological methods. However, when given optimal conditions they can return to normal “culturable” state.

An obvious implication of this phenomenon is that routine examinations of environmental samples for these pathogens can be negative, while virulent bacteria are in fact present.

Epidemiology and risk assessment

Historically cholera is an illness of the poor and undernourished, but this is to some extend due to low standards of hygiene. In the case of cholera, water and fecal contamination of water is of major importance in spread of the disease, but food is becoming increasingly important.

A wide variety of foods has been involved in transmission of cholera, including soft drinks, fruits and vegetables, milk, locally brewed beer as well as millet gruel (Varnam and Evans 1991). However, raw, uncooked, or cross-contaminated cooked shellfish has been established as the major vehicle for V. cholerae 01 and non-01 (Morris and Black 1985). Outbreaks of V. parahaemolyticus has most often been associated with cross-contamination or time/temperature abuse of cooked seafood. An exception is Japan, where raw finfish is the most common vehicle of infection with V. parahaemolyticus. For all other vibrios, consumption of raw shellfish, especially oysters, is the major cause of infection.

An important aspect is the remarkable growth rate exhibited by vibrios in raw fish, even at reduced temperatures. This allows relatively low initial numbers to increase dramatically under improper conditions of harvesting, processing, distribution and storage.

Disease control

Inadequate sanitation and lack of safe water are major causes of cholera epidemics. Therefore cholera can only be reliably prevented by ensuring that all populations have access to adequate excreta disposal systems and safe drinking water. Following the recent outbreak of cholera in South and Central America, WHO (1992) has issued the following recommendations on water supply and sanitation for prevention and control of cholera:

Water supply - WHO recommendations:

1. Drinking water should be adequately disinfected; procedures for disinfection in distribution systems and rural water systems should be improved.

2. Tablets releasing chlorine or iodine may be distributed to the population with instructions on their use.

3. Where chemical treatment of water is not possible, health educators should stress that water for drinking (as well as for washing of hands and utensils) should be boiled before use.

4. Water quality control should be strengthened by intensifying the surveillance and control of residual chlorine, and the conduct and analysis of bacteriological tests, in different points in production and distribution systems.

Sanitation - WHO recommendations:

1. Quality control in sewage treatment plants should be strengthened.

2. The use of treated waste water for irrigation should be carefully controlled, following national and international guidelines.

3. Large-scale chemical treatment of waste water is very rarely justified, even in emergencies, because of the high cost, uncertain effect, and possible adverse impact on the environment and health.

4. Health education should emphasize the safe disposal of human faeces:

• All family members should use a latrine or toilet that is regularly cleaned and disinfected; and

• Faeces of infants and children should be disposed of rapidly in a latrine or toilet, or by burying them.

Vibrios are easily destroyed by heat. Thus proper cooking is sufficient to eliminate most vibrios. However Blake et al. (1980) found V. cholerae 01 to survive boiling for up to 8 min. and steaming for up to 25 min. in naturally contaminated crabs. Thus the commercial practice of heat-shocking oysters in boiling water to facilitate opening is not enough to ensure safety.

At suitable temperatures growth of vibrios can be very rapid. Generation times as short as 8–9 min. have been observed under optimal conditions (37°C). At lower temperatures growth rates are reduced, but it was reported by Bradshaw et al. (1984) that initial concentrations of 10² cfu/g V. parahaemolyticus in homogenized shrimp increased to 10⁸ cfu/g after 24 h at 25°C. These results demonstrate that proper refrigeration is essential in controlling such extravagant growth.

Low temperature storage has been proposed as a means of eliminating pathogenic vibrios from food. However, this method is not of sufficient reliability for commercial application. Mitscherlich and Marth (1984) have reported the survival times of V. cholerae shown in Table 3.5.

Aeromonas sp.

The genus Aeromonas has been classified with the family Vibrionaceae and contains species pathogenic to animals (fish) and man. In recent years the motile Aeromonas sp., particularly A. hydrophila has received increasing attention as a possible agent of foodborne diarrheal disease. However, the role of Aeromonas as an enteric pathogen is not fully clarified.

Aeromonas is ubiquitous in freshwater environments, but may also be isolated from saline and estuarine waters (KnΦchel 1989). This organism may also be readily isolated from meat, fish and seafood, ice-cream and many other foods as reviewed by KnΦchel (1989). Indeed the organism has been identified as the main spoilage organism of raw meat (Dainty et al. 1983), raw salmon (Gibson 1992) packed in vacuum or modified atmospheres, and fish from warm, tropical waters (Gram et al. 1990, Gorczyca and Pek Poh Len 1985).

Species of Aeromonas produce a wide range of toxins such as cytotoxic enterotoxin, hemolysins and a tetrodotoxin-like sodium channel inhibitor (Varnam and Evans 1991). However, the role of these toxins in producing disease in man is unresolved and currently no method is available for differentiating between apathogenic environmental strains and pathogenic strains. Thus there is no evidence that toxins preformed in food play any role, and the association between eating fish and shellfish and Aeromonas-infection is at best circumstantial (Ahmed 1991).

Some growth limiting factors for Aeromonas are shown in Table 3.2. While minimum growth temperature for clinical strains is about +4°C (Palumbo et al. 1985) environmental strains and food isolates have been shown to grow at 0°C (Walker and Stringer 1987). Aeromonas is very sensitive to acid conditions and to salt and growth is unlikely to be a problem in foods where pH is less than 6.5 and the NaCl content greater than 3.0%.

Plesiomonas sp.

Also the genus Plesiomonas is placed in the family of Vibrionaceae. Like other members of this family is Plesiomonas widespread in nature but mostly associated with water, both fresh water and seawater (Arai et al. 1980). Due to its mesophilic nature (see Table 3.2) there is a marked seasonal variation in the numbers isolated from waters being much higher during warmer periods. Transmission by animals and intestines of fish is common, and it is likely that fish and shellfish is the primary reservoir of Plesiomonas shigelloides (Koburger 1989).

Plesiomonas sp. may cause gastroenteritis with symptoms varying from mild illness of short duration to severe diarrhea (shigella-like or cholera-like). However, it is possible that only a few strains carry virulent characteristics since volunteers ingesting the organism do not always become ill (Herrington et al. 1987). As is the case for Aeromonas, there is currently no way to differentiate pathogenic from non-pathogenic Plesiomonas sp.

Growth limiting factors are presented in Table 3.2.

Listeria sp.

Six species of Listeria are currently recognized, but only three sp. L. monocytogenes, L. ivanovii, and L. seeligeri are associated with disease in humans and/or animals. However, human cases involving L. ivanovii and L. seeligeri are extremely rare with only four reported cases. L. monocytogenes is subdivided into 13 serovars on the basis of somatic (0) and flagellar (H) antigens. This subdivision is of limited value in epidemiological studies since most of the isolates belong to three serotypes. More valuable methods are phage-typing, isoenzyme-typing, and DNA-fingerprinting. The latter one has shown promising results (Facinelli et al. 1988, Bille et al. 1992, Gerner-Smidt and NΦrrung 1992).

L. monocytogenes is widespread in nature. It can be isolated from soil, vegetation, foods including fish and fish products and domestic kitchens as reviewed by Lovett (1989), Ryser and Marth (1991) and Fuchs and Reilly (1992). Most of these environmental strains are probably non-pathogenic.

Other Listeria sp. than L. monocytogenes appear to be more common in tropical areas (Fuchs and Reilly 1992, Karunasagar et al. 1992).

Listeriosis is an infection with the intestines as the point of entry, but the infective dose is not known. Incubation period may vary from one day to several weeks. Virulent strains are capable of multiplying in the macrophages and produce septicemia followed by infection of other organs such as the central nervous system, the heart, the eyes and may invade the fetus of pregnant women. In healthy adults, listeriosis usually never develops beyond the primary enteric phase, which may be symptom-free or having only mild “flu-like” symptoms. Listeriosis is of particular risk and can be lethal for foetuses, pregnant women, neonates and immuno-compromised persons.

Epidemiology and risk assessment

Dairy products (milk, cheese, ice-cream, cream butter) have all been implicated in outbreaks of listeriosis. Also salads and vegetables have been involved. Contaminated food is increasingly recognized as an important vehicle of L. monocytogenes. Frequent isolations from seafood (Weagant et al. 1989, RΦrvik and Yndestad 1991) and the demonstration of growth potential in chilled (+4°C) smoked salmon (Ben Embarek and Huss 1992, Guyer and Jemmi 1991, RΦrvik et al. 1991, Fuchs and Reilly 1992) are evidence that seafood may be important in the transmission of Listeria monocytogenes. However, so far there has only been two documented cases of seafood involvement (Facinelli et al. 1989, Frederiksen 1991), and two cases where seafood involvement is suspected (Lennon et al. 1984, Riedo et al. 1990).

Disease control

Currently FDA in the US requires that L. monocytogenes be absent in ready-to-eat seafood products such as crab-meat or smoked fish. This restriction does not apply to raw products that will be cooked before eating (Ahmed 1991). Other countries have similar regulations, which are completely unrealistic, since e.g. cold smoked fish has not been subject to listericidal processing. Due to the ubiquitous nature of L. monocytogenes such products can not be guaranteed free of L. monocytogenes. The FDA is now considering possible changes in their policy (Archer 1992). Products will be classified according to known, established risks. A zero-tolerance will still be maintained for products which have received a listericidal treatment, as well as for products which have been directly implicated in a foodborne outbreak. Low number of L. monocytogenes may then be allowed in other types of products, particularly those in which the organism can be shown to die-off.

There is a general agreement between microbiologists that the presence in our food of low numbers of L. monocytogenes may need to be tolerated. However, Notermans et al. (1992) suggest that a limit of 100 L. monocytogenes/g is reasonable, while Skovgaard (1992) feel that > 10 L. monocytogenes/g is likely to constitute a risk to man - particularly predisposed persons (very old, very young or immuno-suppressed). These quoted figures should be compared to the background level of L. monocytogenes in foods, which is approx. 1–10 L. monocytogenes/g (Skovgaard 1992). This means that little or no growth of L. monocytogenes in foods should be tolerated.

However, the quantitative level of L. monocytogenes contamination on fish products can be maintained at a very low level (< 1–10/g) by proper GMP and factory hygiene. L. monocytogenes is sensitive to sanitizing agents as reviewed by Ryser and Marth (1991). Thus chlorinebased, iodine-based, acid anionic, and quaternary ammonium-type sanitizers are effective against L. monocytogenes at concentrations of 100 ppm, 25–45 ppm, 200 ppm and 100–200 ppm, respectively.

Further disease control with products which have not been subject to listericidal processing rest with control of growth in the products. Some growth limiting factors are listed in Table 3.2. It will be noted that L. monocytogenes is difficult to control in chilled fish products such as, e.g., cold smoked fish. The organism can grow at temperatures down to +1°C, and it is tolerant to NaCl (up to 10% at neutral pH and 25°C). Nitrite is not inhibitory to L. monocytogenes at permitted levels unless there is an interaction with other inhibiting agents (Shahamat et al. 1980). Thus it was demonstrated by Ben Embarek and Huss (1993) that no growth of L. monocytogenes occurred in vacuum packed cold smoked salmon having 5,4% NaCl in water phase and stored at 5°C for 25 days. However, growth was demonstrated in non vacuum packed (Guyer and Jemmi 1991) and vacuum packed (RΦrvik et al. 1991) cold smoked salmon (2.5 – 3.2% NaCl in water phase) and stored at 4°C. Differences in NaCl content and the strains used may explain why different results were seen in these experiments.

Listericidal processing consist primarily of heat treatment. The heat resistance of L. monocytogenes has been the subject of extensive investigation particularly for milk and dairy products as reviewed by Mackey and Bratchell (1989). The thermal death time curve (T DTC) for L. monocytogenes in cod and salmon was studied by Ben Embarek and Huss (1993). The results show a significantly higher heat resistance of L. monocytogenes in salmon fillets compared to cod fillets with D₆₀ being 4.5 min. in salmon and 1.8 min. in cod. The z-values were in both cases ca. 6°C as shown in Figure 3.2 which is very similar to the z-value calculated by Mackey and Bratchell (1989).

Figure 3.2 Heat resistance of L. monocytogenes in cod (open symbols) and salmon fillets (closed symbols). The test organisms were isolated from smoked salmon (squares) and clinical case of listeriosis (triangles) (Ben Embarek and Huss 1993).

3.1.2. Non-indigenous bacteria (Group 2)

Some of the growth requirements of group 2 organisms are listed in Table 3.6.

Salmonella sp.

Salmonella are members of the family Enterobacteriaceae and occur in more than 2000 serovars. These mesophilic organisms are distributed geographically all over the world, but principally occurring in the gut of man and animals and in environments polluted with human or animal excreta. Survival in water depends on many parameters such as biological (interaction with other bacteria) and physical factors (temperature). It has been demonstrated by Rhodes and Kator (1988) that both E. coli and Salmonella sp. can multiply and survive in the estuarine environment for weeks, while Jiménez et al. (1989) presented similar results on survival in tropical freshwater environments.

The principal symptoms of salmonellosis (non-typhoid infections) are non-bloody diarrhea, abdominal pain, fever, nausea, vomiting which generally appear 12–36 hours after ingestion. However, symptoms may vary considerably from grave typhoid like illness to asymptomatic infection. The disease may also proceed to more serious complications. The infective dose in healthy people varies according to serovars, foods involved and susceptibility of the individuals. There is evidence for a minimum infection dose (M.I.D.) of as little as 20 cells (Varnam and Evans 1991), while other studies have consistently indicated > 10⁶ cells.

Epidemiology and risk assessment

Salmonella is occurring commonly in domestic animals and birds and many are asymptomatic Salmonella-excreters. Raw meat and poultry are therefore often contaminated with this organism. Numerous surveys have been conducted as reviewed by D'Aoust (1989), showing that incidence varies according to species, agricultural- and processing practices. Between 50– 100% of all samples of chicken carcasses are positive in most industrialized countries with intensively reared poultry, but also in other meats the contamination may be near to 100%. Also the contamination of raw milk, eggs and egg products with Salmonella is a long standing well known problem.

Contamination of shellfish with Salmonella due to growth in polluted waters has been a problem in many parts of the world. In a recent review by Reilly et al. (1992), evidence is presented that farmed tropical shrimps frequently contain Salmonella. However, it has also demonstrated that Salmonella in aquaculture shrimp products originate from the environment rather than as a result of poor standards of hygiene, sanitation, and poultry manure as feed.

Most literature reports indicate that seafood is a much less common vehicle for Salmonella than other foods, and fish and shellfish are responsible for only a small proportion of total number of Salmonella cases reported in U.S. and elsewhere (Ahmed 1991). Most prawns and shrimps are cooked prior to consumption and these products therefore pose minimal health risks to the consumer except by cross contamination in kitchens.

This is borne out by epidemiological evidence presented by Ahmed (1991), reporting on 7 outbreaks of seafoodborne salmonellosis in USA in the period 1978–1987. Three of these outbreaks were due to contaminated shellfish including 2 outbreaks after consumption of raw oysters harvested from sewage-polluted waters.

Shigella sp.

The genus Shigella is also a member of the Enterobacteriaceae and consists of 4 distinct species. This genus is specific host-adapted to humans and higher primates, and its presence in the environment is associated with fecal contamination. Shigella strains have been reported to survive for up to 6 months in water (Wachsmuth and Morris 1989).

Shigella is the cause of shigellosis (earlier name was bacillary dysentery), which is an infection of the gut. Symptoms vary from asymptomatic infection or mild diarrhea to dysentery, characterized by bloody stools, mucus secretion, dehydration, high fever and severe abdominal cramps. The incubation period for shigellosis is 1–7 days and symptoms may persist for 10–14 days or longer. Death in adults is rare, but the disease in children can be severe. In tropical countries with low standards of nutrition, shigella diarrhea accounts for the death of at least 500.000 children every year (Guerrant 1985).

Epidemiology and risk assessment

The great majority of cases of shigellosis is caused by direct person-to-person transmission of the bacteria via the oral-faecal route. Also waterborne transmission is important, especially where hygiene standards are low.

However, food, including seafood (shrimp-cocktail, tuna salads) have also been the cause of a number of outbreaks of shigellosis. This has nearly always been as a result from contamination of raw or previously cooked foods during preparation by an infected, asymptomatic carrier with poor personal hygiene.

Escherichia coli

E. coli is the most common aerobic organism in the intestinal tract of man and warm blooded animals. Generally the E. coli strains that colonize the gastrointestinal tract are harmless commensals, or they play an important role in maintaining intestinal physiology. However, within the specie there are at least 4 types of pathogenic strains:

1. enteropathogenic E. coli (EPEC)

2. enterotoxigenic E. coli (ETEC)

3. enteroinvasive E. coli (EIEC), shiga-dysentery-like E. coli

4. enterohaemorrhagic E. coli / (EHEC) / verocytoxin producing E. coli (VTEC) or E. coli 0157:H7

Serotyping as well as phage typing and genetic methods are used in epidemiological studies to separate among the various E. coli types, but there are no specific phenotypic marker to separate between pathogenic and non pathogenic strains. However, some atypical properties such as being lactose-negative or failure to produce indole at 44°C are more common between the pathogenic strains (Varnam and Evans 1991). VTEC do not grow at all on selective media at 44°C.

Clearly E. coli may be isolated in environments polluted by faecal material or sewage, and the organism can multiply and survive for a long time in this environment (Rhodes and Kator 1988, Jiménez et al. 1989). However, recently it has been demonstrated that E. coli also can be found in unpolluted warm tropical waters, where it can survive indefinitely (Hazen 1988, Fujioka et al. 1988, Toranzos et al. 1988).

Pathogenic E. coli strains are producing diseases of the gut which may vary in severity from extremely mild to severe and possibly life-threatening depending on a number of factors such as type of pathogenic strains, susceptibility of victim and degree of exposure.

Epidemiology and risk assessment

There is no indication that seafood is an important source of E. coli infection (Ahmed 1991). Most infections appear to be related to contamination of water or handling of food under unhygienic conditions.

Control of Enterobacteriaceae

The Enterobacteriaceae, (Salmonella, Shigella, E. coli) are all occurring on fish products as a result of contamination from the animal/human reservoir. This contamination has normally been associated with fecal contamination or pollution of natural waters or water environments, where these organisms may survive for a long time (months) or through direct contamination of products during processing.

Good personal hygiene and health education of food handlers are therefore essential in the control of diseases caused by Enterobacteriaceae. Proper treatment (e.g. chlorination) of water and sanitary disposal of sewage are also essential parts in a control programme.

Risk of infection with Enterobacteriaceae can be minimized or eliminated by proper cooking before consumption. It is well established that the heat resistance of Salmonella is low, but also that it varies considerably with a_w and with the nature of solutes in the heating menstruum (D'Aoust 1989). Thus a markedly increased heat resistance has been recorded at low a_w. Examples of D-values in high a_w foods are quoted in Table 3.6 as well as other physical factors limiting the growth of Enterobacteriaceae. Thus the growth is generally inhibited in the presence of 4–5% NaCl. Increased inhibition is seen at low temperature or reduced pH. The limiting water activity (a_w) for Salmonella in broth cultures are found to be 0.94 (Marshall et al. 1971).

Growth limiting factors for Shigella and some pathogenic E. coli are of no importance due to the low infective dose required to produce disease.

Current levels of Salmonella in various foods and increasing trends in human infections and foodborne outbreaks (D'Aoust 1989) underline that bacteriological testing and stringent bacteriological standard (zero tolerance limits) of most foods are insufficient measures in the control of salmonellosis. Even the microbial quality of harvest water appears not to be a good predictor of Salmonella contamination, because oysters removed from closed and open beds had same level of contamination (4%) and no correlation was observed between the presence of E. coli and Salmonella (D'Aoust et al. 1980).

Staphylococcus aureus

The staphylococci are ubiquitous organisms and can be found in water, air, dust, milk, sewage, floors, surfaces, all articles that come into contact with man and survive very well in the environment. However, the main reservoir and habitat is the animal/human nose, throat and skin. The human carrier rate may be up to 60% of healthy individuals with an average of 25–30% of the population being positive for enterotoxin-producing strains (Ahmed 1991).

The disease caused by S. aureus is an intoxication. Common symptoms, which may appear within 2–4 hours of consumption of contaminated foods are nausea, vomiting and sometimes diarrhea. Symptoms usually persist for no more than 24 hours, but in severe cases, dehydration can lead to shock and collapse.

Epidemiology and risk assessment

Seafood may be contaminated with Staphylococcus via infected food handlers or from the environment. More often the contamination is from an individual with an infection on hands or with a cold or sore throat.

S. aureus is mesophilic with a minimum growth temperature of 10°C, but higher temperaturesare required for toxin production (> 15°C). In contrast to the Enterobacteriaceae, but in common with L. monocytogenes, S. aureus is halotolerant and able to grow at water activities as low as 0.86. Minimum pH for growth is 4.5. The above minimum requirements are related to growth in laboratory media, when other factors are optimal. This is not always the case in food, where several limiting factors may be acting in combination. It should also be emphasized that staphylococci are poor competitors and do not grow well in the presence of other microorganisms. Thus the presence of staphylococci in raw, naturally contaminated food is of little significance. In contrast rapid growth and toxin production can take place in precooked seafood (shrimp) if recontaminated with S. aureus and time/ temperature conditions allow for growth.

S. aureus produces a number of enterotoxins, when growing in the food. These toxins are generally very resistant to proteolytic enzymes and heat. There have been no outbreaks reported from foods that have undergone normal canning procedures, but the heat applied in pasteurization and normal household cooking is not sufficient to destroy the toxin.

Disease control

Good sanitary conditions and temperature control is necessary to avoid contamination, growth and toxin production - particularly in precooked seafood.

3.2. VIRUS

The incidence of food-borne outbreaks of viral gastroenteritis is still unknown, but some authors believe that they are quite common. Progress has been slow in studying the viruses that infect the human gut and little is known about many of the important characteristics of enteric viruses. Cultivation of some virus (e.g. Hepatitis A virus, HAV) is now possible, but reliable methods for detection of viruses in food is not available. However, techniques based on molecular biology such as RNA/DNA-probes and PCR (polymerase chain reaction) routines are rapidly being developed.

Viral disease transmission to human via consumption of seafood has been known since the 1950's (Roos 1956), and human enteric viruses appear to be the major cause of shellfish-associated disease. Presently there are more than 100 known enteric viruses which are excreted in human faeces and find their way into domestic sewage. However, only a few have shown to cause seafood-associated illness according to Kilgen and Cole (1991).

These are: Hepatitis - type A (HAV)
Norwalk virus (small, round structured)
Snow Mountain Agent
Calicivirus
Astrovirus
Non-A and Non-B.

Viruses are inert outside the living host-cell, but they survive. This means that they do not replicate in water or seafood irrespective of time, temperature or other physical conditions. Their presence on seafood is purely as a result of contamination either via infected food handlers or via polluted water. Shellfish which are filter-feeders tend to concentrate virus from the water in which they are growing. Large amounts of water are passing through active shellfish (up to 1,500 1/day/oyster according to Gerba and Goyal (1978)) which means that the concentration of virus in the shellfish is much higher than in the surrounding water.

Epidemiology and risk assessment

The infective dose of viruses is probably much smaller than that of bacteria for causing foodborne disease (Cliver 1988). The minimum infection dose of some enteric viruses for man is close to the minimum dose detectable in laboratory assay systems using cell cultures (Ward and Akin 1983).

The animal/human bodies are the sources of enteric viruses. The viruses are found in large quantities in the faeces of infected persons a few days to several weeks after ingestion/ infection depending on the virus. Direct or indirect faecal contamination is the most common source of contamination of food.

The list of food vehicles in outbreaks of viral diseases is dominated by bivalve molluscs. However, another important vehicle involves ready to eat food prepared by infected food handlers. The available data show that almost any food that comes into contact with human hands and does not subsequently receive a substantial heat treatment, may transmit these viruses.

With only few exceptions, all reported cases of seafood-associated viral infections have been from consumption of raw or improperly cooked molluscan shellfish (Kilgen and Cole 1991). However, there is clear evidence that HAV has been transmitted by unsanitary practices during processing, distribution or food handling (Ahmed 1991). These seafood associated illnesses are very common. Each year 20,000 to 30,000 cases are reported to the Center of Disease Control (CDC) in U.S. (Ahmed 1991), and one of the largest outbreaks of foodborne illness ever reported, is the outbreak of hepatitis involving 290,000 cases in China in 1988. The investigation revealed that the source and mode of transmission were the consumption of contaminated and inadequately cooked clams (Tang et al. 1991).

The survival of viruses in the environment and in food is dependent on a number of factors such as temperature,salinity, solar radiation, presence of organic solids as reviewed by Gerba (1988). Thus, enteric viruses are able to survive for several months in seawater at temperatures < 10°C, which is much longer than e.g. coliform bacteria (Melnick and Gerba 1980). Thus,there is little or no correlation between presence of virus and the usually applied indicator bacteria for faecal pollution. All enteric viruses are also resistant to acid pH, proteolytic enzymes and bile salts in the gut. Hepatitis type A virus, being one of the more heat stable viruses, has an inactivation time of 10 min at 60°C (Eyles 1989). Thus virus is able to survive some commonly used culinary preparations (steaming, frying). Enteric viruses are also resistant to some common disinfectants (e.g. phenolics, quaternary ammonium compounds, ethanol) while the halogens (e.g. chlorine, iodine) inactivate enteric viruses in water and on clean surfaces. Ozone is highly effective in clean water (quoted after Eyles 1989).

Disease control

Prevention of foodborne viral disease relies on measures to prevent direct or indirect fecal contamination of food that will not receive a virucidal treatment before consumption.

Bivalve shellfish are fit for human consumption if harvested in pollution free waters or alternatively rendered fit by depuration in clean seawater or by cooking. However, there are considerable problems in such a programme:

• Monitoring of harvesting-areas has been based on bacterial indicators of pollution, which are known to be unreliable predictors of viral contamination (Richards 1985, Cliver 1988).

• Depuration technology may be inadequate on some occasions for removal of virus from shellfish (Eyles 1986, Gerba 1988) and there is no practical test to indicate that shellfish have been depurated effectively.

Contamination by food handlers can be prevented by good personal hygiene and health education as mentioned for control with Enterobacteriaceae. Food handlers must not handle food while suffering from intestinal infections and for at least 48 h after symptoms have disappeared. In cases of doubt, disposable gloves should be worn in critical operations, as viruses are difficult to remove from hands by washing and are resistant to many skin disinfectants (Eyles 1989).

3.3. BIOTOXINS

Marine biotoxins are responsible for a substantial number of seafood borne diseases. The toxins which are known are shown in Table 3.7.

The toxins and the diseases they can provoke have been described and reviewed by Taylor (1988), Hall (1991), WHO (1984a, 1989) and Todd (1993), which should be consulted for detailed information. Some of the more important aspects are discussed below.

Tetrodotoxin

Unlike all other biotoxins accumulating in the live fish or shellfish tetrodotoxin is not produced by algae. The precise mechanism in production of this very potent toxin is not clear, but apparently quite commonly occurring symbiont bacteria are involved (Noguchi et al. 1987, Matsui et al. 1989).

Tetrodotoxin is mainly found in the liver, ovaries and intestines in various species of pufferfish, the most toxic being members of the family Tetraodontidae, but not all species in this family contain the toxin. The muscle tissue of the toxic fish is normally free of toxin, but there are exceptions. Pufferfish poisoning causes neurological symptoms 10–45 minutes after ingestion. Symptoms are tingling sensation in face and extremities, paralysis, respiratory symptoms and cardiovascular collapse. In fatal cases death takes place within 6 hours.

Ciguatera

Ciguatera poisoning results from the ingestion of fish that have become toxic by feeding on toxic dinoflagellates, which are microscopic marine planktonic algae. The principal source is the benthic dinoflagellate Gambierdiscus toxicus, which is living around coral reefs closely attached to macroalgae. Increased production of toxic dinoflagellates are seen when reefs are disturbed (hurricanes, blasting of reefs etc.). More than 400 species of fish, all found in tropical or warm waters, have been reported to have caused ciguatera as shown in Figure 3.3 (Halstead 1978). The toxin accumulates in fish that feed on the toxic algae or larger carnivores that prey on these herbivores. Toxin can be detected in gut, liver and muscle tissue by means of mouse-assay and chromatography. Some fish may be able to clear the toxin from their systems (Taylor 1988).

Although the reported incidence of ciguatera poisoning is low (Taylor 1988), it has been estimated that the world-wide incidence may be in the order of 50,000 cases/year (Ragelis 1984). The clinical picture varies but onset time is a few hours after ingestion of toxin. Gastrointestinal and neurological systems are affected (vomiting, diarrhea, tingling sensation, ataxia, weakness). Duration of illness may be 2–3 days but some may also persist for weeks or even years in severe cases. Death results from circulatory collapse. Halstead (1978) has reported a case-fatality rate of about 12%.

Paralytical shellfish poisoning (PSP)

Intoxication after consumption of shellfish is a syndrome that has been known for centuries, the most common being paralytic shellfish poisoning (PSP). PSP is caused by a group of toxins (saxitoxins and derivatives) produced by dinoflagellates of the genera Alexandrium, Gymnodinium and Pyrodinium.

Historically, PSP has been associated with the blooming of dinoflagellates (>10⁶ cells/litre), which may cause a reddish or a yellowish discolouration of the water. However, water discolouration may be caused by proliferation of many types of planktonic species which are not always toxic and not all toxic algae blooms are coloured.

Figure 3.3. World distribution of outbreaks of paralytic shellfish poisoning (black spots) and ciguatera (shaded area). Data from WHO (1984a), Halstead and Schantz (1984) and Lupin (1992).

The dinoflagellates bloom as afunction of water temperature, light, salinity, presence of nutrients and other environmental conditions. However, the precise nature of factors eliciting a toxic clone is unknown. Water temperature must be > 5–8°C for blooms to occur. If temperatures decrease to below 4°C, the dinoflagellates will survive as cysts buried in the upper layers of the sediments. The worldwide occurrence of PSP is shown in Figure 3.3.

Mussels, clams, cockles and scallops that have fed on toxic dinoflagellates retain the toxin for varying periods of time depending on the shellfish. Some clear the toxin very quickly and are only toxic during the actual bloom, others retain the toxin for a long time, even years (Schantz 1984).

PSP is a neurological disorder, and the symptoms, include tingling, burning and numbness of lips and fingertips, ataxia, drowsiness, incoherent speech. In severe cases death occurs due to respiratory paralysis. Symptoms develop within 0.5–2 h of a meal and victims who survive more than 12 h generally recover.

Diarrhetic shellfish poisoning (DSP)

Thousands of cases of gastrointestinal disorders caused by diarrhetic shellfish poisoning(DSP) have been reported in Europe, Japan and Chile (WHO 1984a). The causative dinoflagellates which produce the toxins are within the genus Dinophysis and Aurocentrum. These dinoflagellates are widespread which means that this illness could also occur in other parts of the world. At least 7 toxins have been identified, including okadoic acid. Onset of disease is within half an hour to a few hours following consumption of shellfish which have been feeding on toxic algae. Symptoms are gastrointestinal disorder (diarrhea, vomiting, abdominal pain) and victims recover within 3–4 days. No fatalities have ever been observed.

Neurotoxic shellfish poisoning(NSP)

Neurotoxic shellfish poisoning (NSP) has been described in people who consumed bivalves that have been exposed to “red tides” of the dinoflagellate (Ptychodiscus breve). The disease has been limited to the Gulf of Mexico and areas off the coast of Florida. Brevetoxins are highly lethal to fish and red tides of this dinoflagellate is also associated with massive fish kills.

The symptoms of NSP resembles PSP except that paralysis does not occur. NSP is seldom fatal.

Amnesic shellfish poisoning (ASP)

Amnesic shellfish poisoning (ASP) has only recently been identified (Todd 1990, Addison and Stewart 1989). The intoxication is due to domoic acid, an amino acid produced by the diatom Nitzschia pungens. The first reported incidence of ASP occurred in the winter of 1987/88 in eastern Canada, where over 150 people were affected and 4 deaths occurred after consumption of cultured blue mussels.

The symptoms of ASP vary greatly from slight nausea and vomiting to loss of equilibrium and central neural deficits including confusion and memory loss. The short term memory loss seems to be permanent in surviving victims, thus the term amnesic shellfish poisoning.

Control of disease caused by biotoxins

The control of marine biotoxins is difficult and disease cannot be entirely prevented. The toxins are all of non-protein nature and extremely stable (Gill et al. 1985). Thus cooking, smoking, drying, salting does not destroy them, and one cannot tell from the appearance of fish or shellfish flesh whether it is toxic.

The major preventive measure is inspection and sampling from fishing areas and shellfish beds, and analysis for toxins. The mouse bioassay is often used for this purpose and confirmatory HPLC is done if death occurs after 15 min. If high levels of toxin are found, commercial harvesting is halted. It seems unlikely that it will ever be possible to control phytoplankton composition in growing areas, eliminating toxigenic species, and there is no reliable way to forecast, when a particular phytoplankton will grow and thus no way to predict blooming of toxigenic species (Hall 1991).

Removal of toxin by depuration techniques may have some potential, but the process is very slow and costly. There is also a risk that a small number of individuals decline to open and pump clean water through the system and therefore retain their original level of toxicity (Hall 1991).

To be effective, the monitoring requires reliable sampling plans and efficient means of detection of the toxins. Reliable chemical methods for detection of all toxins are at present available and should be developed. The sampling plan must take into consideration that toxicity of shellfish can increase from negligible to lethal levels in less than one week or even less than 24 h for blue mussels. Also the toxicity can vary within a growing location for shellfish according to geography, water currents and tidal activity.

The present situation regarding tolerances and methods of analysis to be used in a monitoring programme is shown in Table 3.8.

3.4. BIOGENIC AMINES (HISTAMINE POISONING)

Histamine poisoning is a chemical intoxication following the ingestion of foods that contain high levels of histamine. Historically this poisoning was called scombroid fish poisoning because of the frequent association with scombroid fishes including tuna and mackerel.

Histamine poisoning is a world-wide problem occurring in countries where consumers ingest fish containing high levels of histamine. It is a mild disease; incubation period is very short (few minutes to few hours) and duration of illness is short ( few hours). The most common symptoms are cutaneous such as facial flushing, urticaria, edema, but also the gastrointestinal tract may be affected (nausea, vomiting, diarrhea) as well as neurological involvement (headache, tingling, burning sensation in the mouth).

Histamine is formed in the fish post mortem by bacterial decarboxylation of the amino acid histidine as shown in Figure 3.4. The fish frequently involved are those with natural high content of histidine such as those belonging to the family Scombridae but also non-scombroid fish such as Clupeidae and mahi-mahi may be involved in histamine poisoning.

Figure 3.4. Chemical structure of histamine (photo: Pan and James 1985)

The histamine-producing bacteria are certain Enterobacteriaceae, some Vibrio sp. a few Clostridium and Lactobacillus sp. The most potent histamine producers are Morganella morganii, Klebsiella pneumoniae and Hafnia alvei(Stratten and Taylor 1991). These bacteria can be found on most fish, probably as a result of post-harvest contamination. They grow well at 10°C but at 5°C growth is greatly retarded and no histamine was produced by M. morganii when temperatures were <5°C at all times (Klausen and Huss 1987). However, large amounts of histamine were formed by M.morganii at low temperatures (0–5°C) following storage for up to 24 h at high temperatures (10–25°C) even though bacterial growth did not take place at 5°C and below.

Many studies agree that histamine producing bacteria are mesophilic. However, Ababouch et al. (1991) found considerable histamine production in sardines at temperatures <5°C, and van Spreekens (1987) has reported on histamine production by Photobacterium sp. which are also able to grow at temperatures <5°C.

The principal histamine producing bacteria M. morganii grow best at neutral pH, but they can grow in the pH range 4.7–8.1. The organism is not very resistant to NaCl, but at otherwise optimal conditions growth can take place in up to 5% NaCl. Thus histamine production by this organism is only a problem in very lightly salted fish products.

It should be emphasized that once the histamine has been produced in the fish, the risk of provoking disease is very high. Histamine is very resistant to heat, so even if the fish is cooked, canned or otherwise heat-treated before consumption, the histamine is not destroyed.

The evidence that histamine is causing disease is mostly circumstantial. High levels of histamine has consistently been found in samples implicated in outbreaks, and the symptoms noted in outbreaks are consistent with histamine as the causative agents. However, high intake of histamine does not always result in disease, even when “hazard action level” (50 mg/100 g for tunafish) is exceeded.

The human body will tolerate a certain amount of histamine without any reaction. The ingested histamine will be detoxified in the intestinal tract by at least 2 enzymes, the diamine oxidase (DAO) and histamine N-methyltransferase (HMT) (Taylor 1986). This protective mechanism can be eliminated if intake of histamine and/or other biogenic amines is very high, or if the enzymes are blocked by other compounds as shown in Figure 3.5.

Other biogenic amines such as cadaverine and putrescine which are known to occur in spoiled fish may therefore act as potentiators of histamine toxicity. Presumably inhibition of intestinal histamine catabolism will result in greater transport of histamine across cellular membranes and into the blood circulation.

Control of disease caused by biogenic amines

Low temperature storage and holding of fish at all times is the most effective preventive measure. All studies seem to agree that storage at 0°C or very near to 0°C limits histamine formation in fish to negligible levels.

Several countries have adapted regulations governing the maximum allowable levels of histamine in fish. Examples are shown in Table 3.9.

Figure 3.5. The disease concept of food-induced histaminosis (after Sattler and Lorenz 1990)

3.5. PARASITES

The presence of parasites in fish is very common, but most of them are of little concern with regard to economics or public health. Reviews have been published by Healy and Juranek (1979), Higashi (1985) and Olson (1987).

However, more than 50 species of helminth parasites from fish and shellfish are known to cause disease in man. Most are rare and involve only slight to moderate injury but some pose serious potential health risk. The most important are listed in Table 3.10.

All the parasitic helminths have complicated life cycles. They do not spread directly from fish to fish but must pass through a number of intermediate hosts in their development. Very often sea-snails or crustaceans are involved as first intermediate host and marine fish as second intermediate host, while the sexually mature parasite is found in mammals as the final host. In between these hosts, one or more free living stages may occur. Infection of human may be part of this life cycle or it may be a side track causing disruption of the life cycle as shown in Figure 3.6.

Figure 3.6. Life cycle of Anisakis simplex.

Nematodes

Round worms or nematodes are common and found in marine fish all over the world. The anisakis nematodes A. simplex and P. dicipiens commonly known as the herring worm and the cod worm have been intensively studied. They are typical round worms, 1–6 cm long, and if live worms are ingested by humans they may penetrate into the wall of the gastrointestinal tract and cause an acute inflammation (“herring worm disease”). The complete life cycle of Anisakis sp. is shown in Figure 3.6.

A number of other nematodes are found in freshwater fish. Gnathostoma sp. are the most important species found in Asia. The final hosts are cats and dogs but humans may be infected. Upon ingestion the larvae migrate from the stomach to various regions, most commonly to subcutaneous sites on the thorax, arms, head and neck, where the worms induce a creeping sensation and edema.

Another nematode of public health importance is Capillaria sp. (e.g. Capillaria philippinensis). The adult worms are gut parasites in piscivorous birds and intermediate hosts are small freshwater fish. Infection in human causes severe diarrhea and possible death attributed to fluid loss. A well known and common nematode in Asia is the Angiostrongylus sp. (e.g.Angiostrongylus cantonensis). The adult worm is found in the lungs of rats and the inter-mediate hosts are snails, freshwater prawns and land crabs. The parasite has been shown to cause meningitis in man (Figure 3.7).

Figure 3.7. Angiostronglid life cycle. The life of Angiostrongylus sp. is depicted. Sexually dioecious nematodes mate and produce eggs that pass with the faeces or hatch in the intestine. A.cantonensis reaches maturity in the lungs and A.costaricensis reaches maturity in the intestine. The larvae migrate in moist places and may invade invertebrates, such as gastropods. Mammals may encounter infective larvae through the consumption of undercooked infected invertebrates or vegetables. In mammals, the larvae penetrate the intestine and migrate in the viscera. A.cantonensis migrates through the subarachnoid space and develops before migrating to the lungs. In humans, larvae do not migrate beyond the brain. A.cantonensis migrates in the viscera, muscles and skin before returning to the intestine of rats. In humans it continues to migrate until it dies. The life cycle of the gnathosto-matids is similar; they seem to infect and migrate in almost any intermediate host, but mature only in one that provides the proper physiological signal (after Brier 1992).

Cestodes

Only few cestodes or tapeworms in man are known to be transmitted by fish. However, the broad fish tapeworm Diphyllobothrium latum is a common human parasite reaching up to 10 m or more in length in the intestinal tract of man. This parasite has a microcrustacean as first intermediate host and freshwater fish are required as second intermediate host (Figure 3.8). The related species (D. pacificum) is transmitted by marine fish and commonly occurs in coastal regions of Peru, Chile and Japan where raw fish preparations (ceviche, sushi and others) are common.

Figure 3.8. Broad fish tapeworm life cycle. The broad fish tapeworms, Diphyllobothrium sp., reach sexual maturity in the intestinal tract of mammals. Eggs may pass in the faeces and develop in water into larvae that hatch and swim freely. If consumed by a copepoda or other suitable crustacean host, the larvae may then become infective for fish that consume the infected crustacean. These larvae then develop into forms that may infect other fishes, where they do not develop further, or mammals, where they may reach sexual maturity (after Brier 1992).

Trematodes

Some of the Trematodes or flukes are extremely common, particularly in Asia. Thus it is estimated that the Clonorchis sinensis (the liver fluke) is infecting more than 20 million people in Asia. In Southern China human clonorchiasis rates can surpass 40% in some regions (Rim 1982). Intermediate hosts are snails and freshwater fish, while dogs, cats, wild animals and humans are final hosts where the fluke live and develop in the bile-ducts in the liver. The predominant problem in transmission is the contamination of snail-infested waters by egg-laden faeces from humans (e.g. use of “night soil” as fertilizers).

Figure 3.9. Liver fluke life cycle. These trematodes reach sexual maturity in the liver of humans and other mammals. Eggs enter the intestine in the bile, are in-corporated into the faeces of the host and if ingested by amollusc, may hatch. The larvae penetrate the tissues through morphologically distinct stages which by asexual reproduction produce free-swimming larvae. The larvae of Clonorchis sinensis can infect only certain fish species, whereas those of Opisthorchis sinensis can infect either fish or molluscan hosts. In these hosts the larvae become infective for mammals that consume raw or undercooked infected intermediate hosts (after Brier 1992).

Two very small flukes (1–2 mm) Metagonimus yokagawai and Heterophyes heterophies differ from Clonorchis by living in the intestines of the final host, causing inflammation, symptoms of diarrhea and abdominal pain. Intermediate hosts are snails and freshwater fish (Figure 3.10).

Figure 3.10. Heterophyid life cycle. Small intestinal flukes mature sexually in the small intestine of humans and other mammals. They mature deep in intestinal crypts, where some eggs may enter the circulatory system and cause cardiac damage. Eggs that exit in the faeces may develop into larvae, which, if consumed by a compatible gastropod host, hatch and penetrate into the snail's tissues where they develop through two morphologically distinct generations. Motile larvae that result leave the snail host and may penetrate into the tissues of a fish host to form the mammalian infective stage. The life cycle may be completed if humans or other mammals consume the infected fish hosts in a raw or undercooked state (after Brier 1992).

The adult oriental lung fluke Paragonimus sp. is 8–12 mm and encapsulated live in cysts in the lungs of man, cats, dogs and pigs and many wild carnivore animals. Snails and crustaceans (freshwater crab) are the intermediate hosts. (Figure 3.11).

Figure 3.11. Lung fluke life cycle. Paragonimus sp. reach sexual maturity in the lungs of humans and other mammals and are usually found in pairs in the alveolar sacs. Eggs are coughed up and expelled by way of the sputum. They are also excreted in faeces. Free-living larvae hatch from the egg under suitably moist conditions. If these larvae encounter a gastropod host they may enter and develop asexually through two distinct morphological forms into free-living larvae, which then penetrate the soft tissues of a crab or crayfish and encapsulate as the mammalian infective stage. Larvae that are consumed by a mammal then penetrate the intestinal wall and migrate through the tissues. In some hosts, migration continues without further development; however, these larvae remain infective to mammals that consume the uncooked hosts. In hosts that provide the proper physiological signal, the larvae migrate to the lungs and mature (after Brier 1992).

Control of disease caused by parasites

All parasites of concern are transmitted to man by eating raw or uncooked fish products. Control measures to reduce the public health problem related to presence of parasites include legislation and surveillance. In principle the problem can be attacked at 3 levels as listed for nematodes by WHO (1989):

1. Avoidance of capture of nematode-infected fish by selecting specific fishing grounds, specific species or specific age groups.

2. Sorting and removal of nematode-infected fish or removal of nematodes from fish, e.g. by hand over a candling table.

3. Application of techniques to kill nematodes in the fish flesh.

Only 2) and 3) are applied in commercial fishery.

Control measures are particularly important for fish products, which are to be eaten raw or uncooked (matjes-herring, marinated fish, lightly salted fish and cold smoked fish, ceviche, sashimi, sushi etc.). Thus many national health regulations e.g. the German ordinance on health requirements for fish and shellfish (German Fish Ordinance 1988) contain specific rules for handling and processing this type of fish in order to make sure that all nematodes are killed (processing for safety). Based on coordinated research in Holland, Germany and Denmark (Huss et al. 1992), the following criteria for safe processing can be given:

Marinated fish:

Safe processing is primarily based on the level of NaCl in the tissue fluid. When the minimum amount of acetic acid (2.5–3.0% in the tissue fluid) is used, the following maximum, survival time of nematodes at various NaCl-levels was found:

Maximum survival times of nematodes should therefore also be minimum holding time of the final product before sale.

Heat treated fish:

All nematodes were killed when heated to 55°C for 1 minute. This means that hot-smoked, pasteurized, sous-vide cooked and other lightly heat treated fish products are safe. However, some normal household cooking traditions may be on the borderline of safety.

Frozen fish:

Freezing to -20°C and maintaining of this temperature for at least 24 hours will kill all nematodes.

The results listed above show that a number of fish products are unsafe. This applies to lightly salted fish products (<5–6% NaCl in water phase) such as matjes-herring, gravad fish, cold smoked fish, lightly salted caviar, ceviche and several other local traditional products. A short period of freezing either of the raw material or the final product therefore must be included in the processing as a means of control of parasites.

3.6. CHEMICALS

Contamination with chemicals figure very low in official statistics as cause of seafood borne diseases (see Table 2.2).

The chemicals contaminants with some potential for toxicity appear to be (Ahmed 1991):

• Inorganic chemicals: antimony, arsenic, cadmium, lead, mercury, selenium, sulfites (used in shrimp processing).

• Organic compounds: polychlorinated biphenyls, dioxins, insecticides (chlorinated hydrocarbons).

• Processing related compounds: nitrosamines and contaminants related to aquaculture (antibiotics, hormones).

A modest concentration of contaminants are ubiquitous in the clean aquatic environment. A few metals such as copper, selenium, iron and zinc are essential nutrients for fish and shellfish. Contamination occurs when there is a statistically significant increase in the mean levels in comparable organisms.

Problems related to chemical contamination of the environment are nearly all man-made. The ocean dumping of hundreds of millions tons of waste material from industrial processing, sludge from sewage treatment plants, draining into the sea of chemicals used in agriculture and raw untreated sewage from large urban populations and industries all participate in contaminating the coastal marine environments or freshwater environments. From here the chemicals find their way into fish and other aquatic organisms. Increasing amounts of chemicals may be found in predatory species as a result of biomagnification, which is the concentration of the chemicals in the higher levels of the food chain. Or they may be there as a result of bioaccumulation, when increasing concentrations of chemicals in the body tissues accumulated over the life span of the individual. In this case, a large (i.e. an older) fish will have a higher content of the chemical concerned than a small (younger) fish of the same species. The presence of chemical contaminants in seafood is therefore highly dependent on geographic location, species and fish size, feeding patterns, solubility of chemicals and their persistence in the environment.

In a recent review on chemical residue concerns in seafood, Price (1992), concluded that risk from chemical contaminants in commercially harvested fish and shellfish is low and not a problem. Risk from chemical residues (mercury, selenium, dioxins, PCPs, kepone, chlordane, dieldrine and DDT) are primarily a concern with sport caught fish and shellfish, caught in coastal waters and (possibly) in highly polluted waters.

Nevertheless, a large section of a committee report concerned with Seafood Safety in U.S. (Ahmed 1991) has been devoted to occurrence of chemical contamination and related health risks. Some of the general conclusions and recommendations from this report are cited below:

• From both natural and human sources, a small proportion of seafood is contaminated with appreciable concentrations of potentially hazardous organic and inorganic chemicals. Some of the risks that may be significant include reproductive effects from PCBs and methylmercury, and carcinogenesis from selected PCB congeners, dioxins, and some chlorinated hydrocarbon pesticides.

• Consumption of some types of contaminated seafood poses enough risk that efforts toward evaluation, education and control of that risk must be improved.

• Present quantitative risk assessment procedures used by government agencies can and should be improved and extended to non-cancer effects.

• Current monitoring and surveillance programs provide an inadequate representation of the presence of contaminants in edible portions of domestic and imported seafood, resulting in serious difficulties in assessing both risks and specific opportunities for control.

• Because of the unevenness of contamination among species and geographic areas, it is feasible to narrowly target control efforts and still achieve meaningful reductions in exposures.

• The data base for evaluating the safety of certain chemicals that find their way into seafood via aquaculture and processing is too weak to support a conclusion that these products are being effectively controlled.

The principal recommendations of the committee are as follows:

• Existing regulations to minimize chemical and biological contamination of the aquatic environment should be strengthened and enforced.

• Existing FDA and state regulations should be strengthened and enforced to reduce the human consumption of aquatic organisms with relatively high contaminant levels (e.g. certain species from the Great Lakes with high levels of PCBs, swordfish and other species with high methylmercury levels).

• Federal agencies should actively support further research to determine the actual risks from the consumption of contaminants associated with seafood and to develop specific approaches for decreasing these risks.

• Increased environmental monitoring should be initiated at the state level, as part of an overall federal exposure management system.

• States should continue to be responsible for site closures, and for issuing health and contamination advisories tailored to the specific consumption habits, reproductive or other special risks, and information sources of specific groups of consumers.

• There should be an expanded program of public education on specific chemical contaminant hazards via governmental agencies and the health professions.

Some examples of maximum residual chemical contaminants in fish per human consumption are shown in Table 3.11.

3.7. SPOILAGE

Fish tissue is characteristic in being rich in protein and non-protein - nitrogen (e.g. amino acids, trimethylamine-oxide (TMAO), creatinine), but low in carbohydrate resulting in a high post mortem p^H (<6.0). Further, the pelagic, fatty fishes have a high content of lipids consisting mainly of triglycerides with long-chain fatty acids which are highly unsaturated. Also the phospholipids are highly unsaturated and these circumstances have important consequences for spoilage processes under aerobic storage conditions.

The condition named “spoilage”is not clearly defined in objective terms. Obvious signs of spoilage are:

• detection of off-odours and off-flavours

• slime formation

• gas production

• discolouration

• changes in texture

and the development of these spoilage conditions in fish and fish products is due to a combination of microbiological, chemical and autolytic phenomena.

Microbiological spoilage

Initial loss of quality of fresh (non-preserved) lean or non-fatty fish species, chilled or not chilled, is caused by autolytic changes, while spoilage is mainly due to the action of bacteria (see Figure 3.12).

The initial flora on fish is very diverse, although most often dominated by Gram-negative psychrotrophic bacteria. Fish caught in tropical areas, may carry a slightly higher load of Gram-positive organisms and enteric bacteria. During storage a characteristic flora develops, but only a part of this flora contribute to spoilage (see Table 3.12). The specific spoilage organisms (SSO) are producers of the metabolites responsible for the off odours and off flavours associated with spoilage.

Figure 3.12. Changes in sensory quality of iced cod (0°C) (after Huss 1988).

Shewanella putrefaciens is typical for the aerobic chill spoilage of many fish from temperate waters and produces trimethylamine (TMA), hydrogen sulphide (H₂S) and other volatile sulphides which give rise to the fishy, sulphidy cabbage like off-odours and - flavours. Similar metabolites are formed by Vibrionaceae and Enterobacteriaceae during spoilage at higher temperatures. During storage in modified atmosphere (CO₂-containing), a psychrophilic Photobacterium producing large amounts of TMA is one of the major spoilage bacteria. Some fresh water fish and many fish from tropical waters are during iced, aerobic storage characterized by a Pseudomonas type of spoilage which is described as fruity, sulphydryl and sickening. Several volatile sulphides (e.g. methylmer-captan (CH₃SH) and dimethylsulphide ((CH₃)₂S), ketones, esters and aldehydes but not hydrogen sulphide are produced by Pseudomonas as are several ketones, esters and aldehydes. For fresh, non-preserved fish, the SSO which have been identified are shown in Table 3.12. Putrefaction or spoilage proceeds very rapidly once the load of SSO exceeds approximately 10⁷ CFU/g.

Microbiological activity is also the cause of spoilage of many preserved fish products stored at temperatures >0°C. However, in most cases the specific spoilage bacteria are not known. The addition of small amounts of salt and acid, as in lightly preserved fish products, changes the dominating microflora to consist mainly of Gram positive bacterial species (Lactic acid bacteria, Brochotrix) and some of these may act as SSO under certain conditions as shown in Table 3.13. However, also some Enterobacteriaceae and Vibrionaceae may act as SSO for these products. In products with low levels of preservation, Shewanella putrefaciens may also play a role.

Also more strongly preserved fish products such as salt cured or fermented products spoil due to the action of certain microorganisms. The dominating flora on these products are Gram positive, halophilic or halotolerant micrococci, yeasts, spore formers, lactic acid bacteria and moulds. A number of SSO are known such as the extremely halophilic, anaerobic Gram negative rods and halophilic yeasts identified by KnΦchel and Huss (1984) as specific spoilage organisms by causing off-odours and -flavours (sulphidy, fruity) in wet salted herring. An extreme halophile spoilage bacteria cause a condition known as “pink”. These bacteria (Halococcus and Halobacterium) cause pink discolouration of salt, brines and salted fish as well as off odours and -flavours normally associated with spoilage (hydrogen sulphide and indole).

Some halophilic moulds (Sporendonema, Oospora) are also classified as spoilers. They do not produce off odours but their presence detracts from the value of the product because of their undesirable appearance.

1) Modified Atmosphere Packaging (CO₂ containing),

2) LAB: Lactic Acid Bacteria

3) Fish caught in tropical waters or fresh waters tend to have a spoilage dominated by Pseudomonas sp.

References: 1) Dalgaard et al. (1993), 2) Gram et al. (1987), 3) Lima dos Santos (1978), 4) Gram et al. (1990), 5) Gorczyca and Pek Poh Len (1985), 6) Donald and Gibson (1992), 7) van Spreekens (1977), 8) Barile et al. (1985), 9) JΦrgensen and Huss (1989).

1) i.e. specific spoilage organisms which have been related to the spoilage of the product2) LAB = Lactic Acid Bacteria

Chemical spoilage (Oxidation)

The most important chemical spoilage processes are changes taking place in the lipid fraction of the fish. Oxidative processes, autoxidation, is a reaction involving only oxygen and unsaturated lipid. At first step leads to formation of hydroperoxides, which are tasteless but can cause brown and yellow discolouration of the fish tissue. The degradation of hydro-peroxides gives rise to formation of aldehydes and ketones as shown in Figure 3.13. These compounds have a strong rancid flavour. Oxidation may be initiated and accelerated by heat, light (especially UV-light) and several organic and inorganic substances (e.g. Cu and Fe). Also a number of antioxidants with the opposite effect are known (alpha-tocopherol, ascorbic acid, citric acid, carotenoids).

Figure 3.13. Basic processes for oxidation of polyunsaturated fatty acids found in fish tissue (after Ackman and Ratnayake 1992).

Autolytic spoilage

Autolytic spoilage or autolytic changes are responsible for early quality loss in fresh fish but contribute very little to spoilage of chilled fish and fish products. An exception from this statement is the rapid development of off-odours and discolourations due to action of gut enzymes in certain ungutted fish. However, in frozen fish the autolytic changes are of great importance. One example is the reduction of trimethylamine-oxide (TMAO), which in chilled fish is a bacterial process with formation of trimethylamine (TMA). In frozen fish, however, bacterial action is inhibited and TMAO is broken down by autolytic enzymes to dimethylamine (DMA) and formaldehyde (FA):

(CH₃)N:O √ (₃)₂ NH + HCHO

The effect of the FA formed in frozen fish is increased denaturation of fish tissue, changes in texture and loss of water binding capacity. Other enzymatic reactions such as formation of free fatty acids are also believed to greatly influence the sensory quality of frozen fish. Autolytic enzymes are active even at -20°C and below, but are proceeding at a much faster rate at high, sub-zero temperatures.

The causes of the various types of spoilage are summarized in Table 3.14.

Control of spoilage

All proteinaceous foods spoil sooner or later, but a number of measures can be taken to reduce spoilage rate. Greatest effect can be obtained by control of storage temperature. As stated, the major cause of spoilage is bacterial, and in the chill temperature range the growth pattern of psychrotrophic spoilage organisms can be described accurately by the square root relation as reviewed by Bremner et al. (1987). Thus when 0°C is used as a reference temperature, the relationship comparing growth (r) at any particular temperature with that at 0°C becomes:

√ _r = 1 + 0.1 × t where t is temperature in °C.

This means, ife.g. storage temperature is 10°C, the growth of spoilage bacteria is 4 times faster than at 0°C (√ _r = 1 + 0.1 × 10, r=4) and shelflife is reduced correspondingly.

Chemical spoilage or development of rancidity can be prevented by rapid catch handling on board and storage of products under anoxic conditions (vacuum packed or modified atmosphere packed). Use of antioxidants may be considered.

The effect of storage temperature on quality of frozen fish is also pronounced and spoilage rate is considerably reduced at temperatures below -20°C.

The effect of hygiene in control of spoilage varies depending on the type of contamination which may take place. Great effort to reduce the general contamination during catch handling on board did not lead to any significant delay in spoilage (Huss et al. 1974) as only a very small part of this general contamination is made up of specific spoilage bacteria. In contrast, hygiene measures to control contamination of fish and fish products with specific spoilage bacteria greatly influences spoilage rate and shelflife (JΦrgensen et al. 1988).