《食品包装技术》（英文版）Chapter 12 Reducing pathogen risks in MAP-prepared produce.pdf_大学文库

232 Novel food packaging techniques during harvesting and manual preparation(human contact) or during machine processing and packaging(contaminated work surfaces/packaging materials/ equipment). Cross-contamination by end-users after pack opening can also By extending shelf-life and protecting product quality, MAP prepared produce systems can provide sufficient time for pathogens to grow to significant numbers on otherwise acceptable fresh foods(Berrang et al, 1989b). The risk of food poisoning is greatest in products eaten raw without any further preparation While the food safety record of these products is good, a comprehensive understanding of the implications of this technology for pathogen survival and growth is required in order to optimise production systems and to inform HACCP protocols. The effects of MAP technology on the survival and growth of non-pathogens and on the interaction between pathogens and non-pathogens also important(Francis and O Beirne, 1998b). Non-pathogens are both potential competitors of pathogens and important indicators of product spoilage While considerable progress has been made in the past decade in our understanding of the safety of these novel and complex food systems there are still significant gaps in knowledge requiring further research 12.2 Measuring pathogen risks A range of pathogens have been isolated from raw produce(Brackett, 1999, Francis et al, 1999) and foodborne infections have been linked to the consumption of raw vegetables and fruits (Tables 12.1 and 12.2). While pathogens have also been isolated from MAP prepared produce(see Table 12. 1) relatively few foodborne infections have been directly linked with this range of oducts. those that have been linked include an outbreak of botulism ultimately linked to an MaP dry coleslaw product( Solomon et al., 1990)and a Salmonella Newport outbreak linked to ready-to-eat salad vegetables(PHLS 2001). There was also an outbreak of shigellosis linked to shredded lettuce (Davis et al., 1988)though exactly how this product was packaged is unclear Increasing consumption of fresh produce in the United States has been paralleled by an increase in produce-linked food poisoning outbreakS(NACMCF, 1999) Contributory factors include the increased range and diversity of products available to consumers and the elimination of seasonality by almost year-round availability of many commodities. This diversity and availability has been achieved by increased globalisation of the produce trade, and has brought with it new food safety risks and challenges. While the main pathogens of concern are still non-proteolytic Clostridium botulinum, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila, there are important emerging threats from viral and protozoan pathogens There are a number of difficulties in estimating the magnitude of the true microbial risk from fresh produce and MAP fresh produce. Studies where samples of produce are examined for the presence of pathogens are, of necessity

during harvesting and manual preparation (human contact) or during machine processing and packaging (contaminated work surfaces/packaging materials/ equipment). Cross-contamination by end-users after pack opening can also occur. By extending shelf-life and protecting product quality, MAP prepared produce systems can provide sufficient time for pathogens to grow to significant numbers on otherwise acceptable fresh foods (Berrang et al., 1989b). The risk of food poisoning is greatest in products eaten raw without any further preparation. While the food safety record of these products is good, a comprehensive understanding of the implications of this technology for pathogen survival and growth is required in order to optimise production systems and to inform HACCP protocols. The effects of MAP technology on the survival and growth of non-pathogens and on the interaction between pathogens and non-pathogens is also important (Francis and O’Beirne, 1998b). Non-pathogens are both potential competitors of pathogens and important indicators of product spoilage. While considerable progress has been made in the past decade in our understanding of the safety of these novel and complex food systems there are still significant gaps in knowledge requiring further research. 12.2 Measuring pathogen risks A range of pathogens have been isolated from raw produce (Brackett, 1999; Francis et al., 1999) and foodborne infections have been linked to the consumption of raw vegetables and fruits (Tables 12.1 and 12.2). While pathogens have also been isolated from MAP prepared produce (see Table 12.1) relatively few foodborne infections have been directly linked with this range of products. Those that have been linked include an outbreak of botulism ultimately linked to an MAP dry coleslaw product (Solomon et al., 1990) and a Salmonella Newport outbreak linked to ready-to-eat salad vegetables (PHLS, 2001). There was also an outbreak of shigellosis linked to shredded lettuce (Davis et al., 1988) though exactly how this product was packaged is unclear. Increasing consumption of fresh produce in the United States has been paralleled by an increase in produce-linked food poisoning outbreaks (NACMCF, 1999). Contributory factors include the increased range and diversity of products available to consumers and the elimination of seasonality by almost year-round availability of many commodities. This diversity and availability has been achieved by increased globalisation of the produce trade, and has brought with it new food safety risks and challenges. While the main pathogens of concern are still non-proteolytic Clostridium botulinum, Listeria monocytogenes, Yersinia entercolitica and Aeromonas hydrophila, there are important emerging threats from viral and protozoan pathogens. There are a number of difficulties in estimating the magnitude of the true microbial risk from fresh produce and MAP fresh produce. Studies where samples of produce are examined for the presence of pathogens are, of necessity, 232 Novel food packaging techniques

Reducing pathogen risks in MAP-prepared produce 233 limited in size and may not accurately reflect global contamination levels. In than those showing their presence, and this may distort the true picture. A recent examination of 127 fresh produce items from the Washington DC area Thunberg et al, 2002)showed low levels of contaminati Salmonella Campylobacter contamination, and seven samples positive for L nocytogenes. On the other hand, food poisoning incidents related to fresh produce may be under-reported. By comparison with those linked to meat and poultry, outbreaks related to produce do not have the same pathogen and product characteristics which assist in recognition, investigation, and reporting (NACMCF, 1999). For example, the short shelf-lives, complex distribution and universal consumption of fresh produce make produce-implicated outbreaks more difficult to pin down. Even when produce is almost certainly implicated, the exact point of contamination is difficult to prove beyond doubt. Of 27 examples of produce-linked food poisoning outbreaks considered by NACMCF, investigators had definitively identified the point of contamination in only two The main pathogens of concern in MAP produce are discussed below, focusing on sources and levels of contamination, and their likely health risk to consumers 12. 2.1 Listeria monocytogenes L. monocytogenes is a Gram-positive rod which causes several diseases in man including meningitis, septicaemia, still-births and abortions(ICMSF, 1996). It is considered ubiquitous in the environment, being isolated from soil, faeces, sewage, silage, manure, water, mud, hay, animal feeds, dust, birds, animals and man(Al-Ghazali and Al-Azawi, 1990, Gunasena et al., 1995, Gray and Killinger, 1966; Nguyen-the and Carlin, 1994; Welshimer, 1968) Contamination of vegetables by L. monocytogenes may occur through agricultural practices, such as irrigation with polluted water or use of contaminated manure(Nguyen-the and Carlin, 1994; Geldreich and Bordner 1971). It may also occur during processing(see Section 12.3.3).L monocytogenes has been isolated from minimally processed vegetables at rates ranging from 0%(Farber et al, 1989, Fenlon et al, 1996, Gohil et al., 1995: Petran et al, 1988)to 44%(Arumugaswamy et al., 1994, Beckers et al., 1989 Doris and Seah, 1995, Harvey and Gilmour, 1993; Mac Gowan et al., 1994 McLauchlin and gilbert, 1990; Sizmur and Walker, 1988; Velani and roberts 1991). In France(Nguyen-the and Carlin, 1994)and Germany(Lund, 1993) levels of>10 CFU/g are unacceptable, while in the UK and USA the organism must be absent in 25g Of particular concern is the organism's ability to grow at refrigeration temperatures; the minimum temperature for growth is reported to be -04C (Walker and Stringer, 1987). It is also facultatively anaerobic, capable of survival/growth under the low O2 concentrations within MA packages of prepared vegetables. While counts generally remain constant at 4C(Farber et al., 1998), they can increase to high numbers at mild abuse temperatures( 8C)

limited in size and may not accurately reflect global contamination levels. In addition, surveys showing the absence of pathogens may receive less attention than those showing their presence, and this may distort the true picture. A recent examination of 127 fresh produce items from the Washington DC area (Thunberg et al., 2002) showed low levels of contamination, no Salmonella or Campylobacter contamination, and seven samples positive for L. monocytogenes. On the other hand, food poisoning incidents related to fresh produce may be under-reported. By comparison with those linked to meat and poultry, outbreaks related to produce do not have the same pathogen and product characteristics which assist in recognition, investigation, and reporting (NACMCF, 1999). For example, the short shelf-lives, complex distribution and universal consumption of fresh produce make produce-implicated outbreaks more difficult to pin down. Even when produce is almost certainly implicated, the exact point of contamination is difficult to prove beyond doubt. Of 27 examples of produce-linked food poisoning outbreaks considered by NACMCF, investigators had definitively identified the point of contamination in only two. The main pathogens of concern in MAP produce are discussed below, focusing on sources and levels of contamination, and their likely health risk to consumers. 12.2.1 Listeria monocytogenes L. monocytogenes is a Gram-positive rod which causes several diseases in man including meningitis, septicaemia, still-births and abortions (ICMSF, 1996). It is considered ubiquitous in the environment, being isolated from soil, faeces, sewage, silage, manure, water, mud, hay, animal feeds, dust, birds, animals and man (Al-Ghazali and Al-Azawi, 1990; Gunasena et al., 1995; Gray and Killinger, 1966; Nguyen-the and Carlin, 1994; Welshimer, 1968). Contamination of vegetables by L. monocytogenes may occur through agricultural practices, such as irrigation with polluted water or use of contaminated manure (Nguyen-the and Carlin, 1994; Geldreich and Bordner, 1971). It may also occur during processing (see Section 12.3.3). L. monocytogenes has been isolated from minimally processed vegetables at rates ranging from 0% (Farber et al., 1989; Fenlon et al., 1996; Gohil et al., 1995; Petran et al., 1988) to 44% (Arumugaswamy et al., 1994; Beckers et al., 1989; Doris and Seah, 1995; Harvey and Gilmour, 1993; MacGowan et al., 1994; McLauchlin and Gilbert, 1990; Sizmur and Walker, 1988; Velani and Roberts, 1991). In France (Nguyen-the and Carlin, 1994) and Germany (Lund, 1993) levels of >102 CFU/g are unacceptable, while in the UK and USA the organism must be absent in 25g. Of particular concern is the organism’s ability to grow at refrigeration temperatures; the minimum temperature for growth is reported to be ÿ0.4ºC (Walker and Stringer, 1987). It is also facultatively anaerobic, capable of survival/growth under the low O2 concentrations within MA packages of prepared vegetables. While counts generally remain constant at 4ºC (Farber et al., 1998), they can increase to high numbers at mild abuse temperatures (8ºC), Reducing pathogen risks in MAP-prepared produce 233

236 Novel food packaging particularly after anti-microbial dipping treatments or within nitrogen flushed packages(Francis and O'Beirne, 1997). However, evidence is emerging that levels of virulence may vary greatly among L. monocytogenes strains, and that some serotypes found in MAP produce may be different(and less virulent) than those isolated in food poisoning outbreaks(Beuchat and Ryu, 1997). Further research is required to determine the significance of different L.monocytogenes strains for human health 12.2.2 Clostridium botulinum 1. botulinum is a member of the genus Clostridium, characterised as Gram- positive, rod-shaped, endospore forming, obligate anaerobes (Varnum and Evans, 1991). The foodborne Clostridia have been comprehensively reviewed by McClane(1997) and Dodds and Austin (1997). Cl. botulinum is divided into numerous sub-divisions, based on the serological specificity of the neurotoxin produced, and physiological differences between strains. Human botulism is normally attributed to sub-species antigenic types A, B, E and occasionally type F. Endospores of Cl. botulinum are ubiquitous, being distributed in soils, aquatic sediments and the digestive tract of animals and bird Vegetables are potentially contaminated during growth, harvesting and processing(Rhodehamel, 1992). Despite their ubiquity, a recent study identified only 0.36% of pre-cut MAP vegetables to be contaminated with Cl. botulinum pores (Lilly et al, 1996). In the case of mushrooms, a much lower incidence of Cl. botulinum was reported(Notermans et al, 1989)than had been reported previously(Hauschild et al, 1978), a change attributed to hygienic improve- ments in growing techniques The possibility of growth and toxin production by Cl. botulinum before obvious spoilage has long been of concern in over-wrapped mushrooms (Sugiyama and Yang, 1975)and in vacuum packaged prepared potatoes (O Beirne and Ballantyne, 1987). In addition, sufficiently anoxic conditions are frequently observed in Ma packages where the respiration rate of the product is not matched by the permeability of the packaging used. Anoxic conditions may also develop within MAP produce where edible coatings are used( Guilbert et al., 1996). Highly permeable or perforated over-wrapping films have been used for fresh mushrooms and low storage temperatures and short shelf-lives have been requirements in prepared potato products (IFST, 1990). In the case of other items of vacuum packaged/MAP prepared produce, the data suggest that spoilage is likely to preceed toxin production(Larson et al., 1997; Petran et al n in production to occur prior to obvious spoilage (Larson et al, 1997). However, there is a report linking a botulism outbreak with coleslaw prepared from a MAP dry coleslaw mix Solomon et al., 1990). The short shelf-lives of retail packs and the good control of temperature/modest storage lives of catering packs are likely to minimise such risks, but there is need for vigilance and further research

particularly after anti-microbial dipping treatments or within nitrogen flushed packages (Francis and O’Beirne, 1997). However, evidence is emerging that levels of virulence may vary greatly among L. monocytogenes strains, and that some serotypes found in MAP produce may be different (and less virulent) than those isolated in food poisoning outbreaks (Beuchat and Ryu, 1997). Further research is required to determine the significance of different L. monocytogenes strains for human health. 12.2.2 Clostridium botulinum Cl. botulinum is a member of the genus Clostridium, characterised as Grampositive, rod-shaped, endospore forming, obligate anaerobes (Varnum and Evans, 1991). The foodborne Clostridia have been comprehensively reviewed by McClane (1997) and Dodds and Austin (1997). Cl. botulinum is divided into numerous sub-divisions, based on the serological specificity of the neurotoxin produced, and physiological differences between strains. Human botulism is normally attributed to sub-species antigenic types A, B, E and occasionally type F. Endospores of Cl. botulinum are ubiquitous, being distributed in soils, aquatic sediments and the digestive tract of animals and birds. Vegetables are potentially contaminated during growth, harvesting and processing (Rhodehamel, 1992). Despite their ubiquity, a recent study identified only 0.36% of pre-cut MAP vegetables to be contaminated with Cl. botulinum spores (Lilly et al., 1996). In the case of mushrooms, a much lower incidence of Cl. botulinum was reported (Notermans et al., 1989) than had been reported previously (Hauschild et al., 1978), a change attributed to hygienic improvements in growing techniques. The possibility of growth and toxin production by Cl. botulinum before obvious spoilage has long been of concern in over-wrapped mushrooms (Sugiyama and Yang, 1975) and in vacuum packaged prepared potatoes (O’Beirne and Ballantyne,1987). In addition, sufficiently anoxic conditions are frequently observed in MA packages where the respiration rate of the product is not matched by the permeability of the packaging used. Anoxic conditions may also develop within MAP produce where edible coatings are used (Guilbert et al., 1996). Highly permeable or perforated over-wrapping films have been used for fresh mushrooms and low storage temperatures and short shelf-lives have been requirements in prepared potato products (IFST, 1990). In the case of other items of vacuum packaged/MAP prepared produce, the data suggest that spoilage is likely to preceed toxin production (Larson et al., 1997; Petran et al., 1995), with a probability of 1 in 105 for toxin production to occur prior to obvious spoilage (Larson et al., 1997). However, there is a report linking a botulism outbreak with coleslaw prepared from a MAP dry coleslaw mix (Solomon et al., 1990). The short shelf-lives of retail packs and the good control of temperature/modest storage lives of catering packs are likely to minimise such risks, but there is need for vigilance and further research. 236 Novel food packaging techniques

Reducing pathogen risks in MAP-prepared produce 237 12.2.3 Escherichia coli 0157: H7 E. coli, type species of the type Enterobacteriaceae genus, Escherichia, is a common inhabitant of the gastrointestinal tract of mammals. Despite the commensal status of the majority of strains, pathogenic ula enterohaemorrhagic E. coli o157: H7. have emerged as foodborne pathogens. Gastroenteritis and haemorrhagic are classical symptoms, while complications including thrombocytopenic purpura and haemolytic uraemic syndrome have been documented(Martin et al., 1986) the latter potentially leading to renal failure and death in 3-5% of juvenile cases armani et al, 1983; Griffin and Tauxe, 1991) The principal reservoir of E. coli o157: H7 is believed to be the bovine gastrointestinal tract (Wells et al., 1991; Doyle et al., 1997).Hence, contamination of meat and other food products with faeces is a significant risk factor. Contamination of, and survival of the organism in natural water sources make these also potential sources in the distribution of infection particularly if untreated water is used to wash produce. The potential for cross- contamination during distribution and domestic storage are also of concern Information regarding contamination rates of MAP prepared vegetables is limited. Recent surveys in the UK and US failed to find this pathogen(FDA 2001) 12.2.4 Aeromonas hydrophila Aeromonas hydrophila is a motile, Gram-negative, rod-shaped bacterium in the family Vibrionaceae. It causes a broad spectrum of infections(septicaemia meningitis, endocarditis)in humans, often in immunocompromised hosts, and Aeromonas spp. have been associated epidemiologically with travellers diarrhoea. Its significance as a human pathogen has been reviewed by Altwegg and Geiss(1989) A hydrophila is considered to be ubiquitous and has been isolated from man ources. The best known sources are treated and untreated water and animals associated with water, such as fish and shellfish (ICMSF, 1996). hazen et al 1978)isolated A. hydrophila from the vast majority of aquatic environments. A hydrophila is also associated with soil Brandi et al., 1996)and with a range of foods including fresh vegetables. Foods in which A. hydrophila was isolated were most likely contaminated by water, soil or animal faeces A hydrophila possesses a number of characteristics of concern in relation to lAP prepared vegetables. It is a psychrotroph; it grows slowly at 0oC, but temperatures of 4-5C will support growth in foods. It is also a facultative anaerobe, capable of growing in atmospheres containing low concentrations of oxygen. Marchetti et al.(1992)isolated high counts (10-10/ hydrophila in commercial MAP prepared vegetable salads. Aeromonas spp were also recovered from green salad, coleslaw(Hudson and De Lacy, 1991). pre-made salad samples(Fricker and Tompsett, 1989), mayonnaise salad samples(Knochel and Jeppesen, 1990)and commercial mixed vegetable salads

12.2.3 Escherichia coli O157:H7 E. coli, type species of the type Enterobacteriaceae genus, Escherichia, is a common inhabitant of the gastrointestinal tract of mammals. Despite the commensal status of the majority of strains, pathogenic strains, particularly enterohaemorrhagic E. coli O157:H7, have emerged as highly significant foodborne pathogens. Gastroenteritis and haemorrhagic colitis are classical symptoms, while complications including thrombocytopenic purpura and haemolytic uraemic syndrome have been documented (Martin et al., 1986), the latter potentially leading to renal failure and death in 3–5% of juvenile cases (Karmali et al., 1983; Griffin and Tauxe, 1991). The principal reservoir of E. coli O157:H7 is believed to be the bovine gastrointestinal tract (Wells et al., 1991; Doyle et al., 1997). Hence, contamination of meat and other food products with faeces is a significant risk factor. Contamination of, and survival of the organism in natural water sources make these also potential sources in the distribution of infection, particularly if untreated water is used to wash produce. The potential for crosscontamination during distribution and domestic storage are also of concern. Information regarding contamination rates of MAP prepared vegetables is limited. Recent surveys in the UK and US failed to find this pathogen (FDA, 2001). 12.2.4 Aeromonas hydrophila Aeromonas hydrophila is a motile, Gram-negative, rod-shaped bacterium in the family Vibrionaceae. It causes a broad spectrum of infections (septicaemia, meningitis, endocarditis) in humans, often in immunocompromised hosts, and Aeromonas spp. have been associated epidemiologically with travellers diarrhoea. Its significance as a human pathogen has been reviewed by Altwegg and Geiss (1989). A. hydrophila is considered to be ubiquitous and has been isolated from many sources. The best known sources are treated and untreated water, and animals associated with water, such as fish and shellfish (ICMSF, 1996). Hazen et al. (1978) isolated A. hydrophila from the vast majority of aquatic environments. A. hydrophila is also associated with soil (Brandi et al., 1996) and with a range of foods including fresh vegetables. Foods in which A. hydrophila was isolated were most likely contaminated by water, soil or animal faeces. A. hydrophila possesses a number of characteristics of concern in relation to MAP prepared vegetables. It is a psychrotroph; it grows slowly at 0ºC, but temperatures of 4–5ºC will support growth in foods. It is also a facultative anaerobe, capable of growing in atmospheres containing low concentrations of oxygen. Marchetti et al. (1992) isolated high counts (103 –106 /g) of A. hydrophila in commercial MAP prepared vegetable salads. Aeromonas spp. were also recovered from green salad, coleslaw (Hudson and De Lacy, 1991), pre-made salad samples (Fricker and Tompsett, 1989), mayonnaise salad samples (Knøchel and Jeppesen, 1990) and commercial mixed vegetable salads Reducing pathogen risks in MAP-prepared produce 237

238 Novel food packaging techniques ( Garcia-Gimeno et al, 1996). By contrast, none of the vegetable samples from shops in Sweden was positive for Aeromonas spp (Krovacek et al, 1992) salmonella, a genus of the family Enterobacteriaceae, are characterised as Gram-negative, rod-shaped bacteria. Pathogenic species include S Typhimurium, S. Enteritidis, S. Heidelberg, S. Saint-paul, and S. Montevideo Salmonellae are mesophiles, with optimum temperatures for growth of 35-43.C The growth rate is substantially reduced at <15C, while the growth of most salmonellae is prevented at <7C. Salmonella are facultatively anaerobic capable of survival in low O2 atmospheres These organisms are abundant in faecal material, sewage and sewage-polluted water; consequently they may contaminate soil and crops with which they come into contact. Sewage sludge may contain high numbers of salmonellae and, if used for agricultural purposes, will disseminate the bacterium. Once introduced into the environment, salmonellae remain viable for months (ICMSF, 1996). Potential contamination from workers who handle produce in the field or in processing plants is of great concern(see Section 12. 4). Salmonellae have not generally been found in MAP produce, though they have been isolated from bean-sprouts(20%)in Malaysia(Arumugaswamy et al., 1994) 12.2.6 Yersinia enterocolitica Y. enterocolitica is currently considered to be the most significant genus member with respect to foodborne disease (Varnum and Evans, 1991) Traditional gastrointestinal symptoms, potentially mediated through the activity of a heat-stable enterotoxin, may develop into suppurative and autoimmune complications (Robins-Browne, 1997). The psychrotrophic status of y. enterocolitica is potentially of great significance with regard to refrigerated MAP prepared produce Y. enterocolitica occupies a broad range of ecosystems including the intestinal tract, birds, flies, fish and a variety of terrestrial and aquatic ecosystems. However, most environmental isolates lack virulence markers and are of doubtful significance for human or animal health(Delmas and idon, 1985). Isolation of Yersinia spp. from raw vegetables has been reported at rates ranging from 3.3%(Tassinari et al., 1994)to 46.1%(Delmas and Idon, 1985) although specific isolation rates of pathogenic Y enterocolitica strains are likely to be significantly lower 12.2.7 Campylobacter jejuni Since their principal identification as human gastrointestinal pathogens in the 1970s(Butzler et al., 1973: Skirrow, 1977) members of the thermophilic campylobacters, e.g. C. jejuni, have emerged as major human gastrointestinal

(Garcı´a-Gimeno et al., 1996). By contrast, none of the vegetable samples from shops in Sweden was positive for Aeromonas spp. (Krovacek et al., 1992). 12.2.5 Salmonella Salmonella, a genus of the family Enterobacteriaceae, are characterised as Gram-negative, rod-shaped bacteria. Pathogenic species include S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Saint-paul, and S. Montevideo. Salmonellae are mesophiles, with optimum temperatures for growth of 35–43ºC. The growth rate is substantially reduced at <15ºC, while the growth of most salmonellae is prevented at <7ºC. Salmonella are facultatively anaerobic, capable of survival in low O2 atmospheres. These organisms are abundant in faecal material, sewage and sewage-polluted water; consequently they may contaminate soil and crops with which they come into contact. Sewage sludge may contain high numbers of salmonellae and, if used for agricultural purposes, will disseminate the bacterium. Once introduced into the environment, salmonellae remain viable for months (ICMSF, 1996). Potential contamination from workers who handle produce in the field or in processing plants is of great concern (see Section 12.4). Salmonellae have not generally been found in MAP produce, though they have been isolated from bean-sprouts (20%) in Malaysia (Arumugaswamy et al., 1994). 12.2.6 Yersinia enterocolitica Y. enterocolitica is currently considered to be the most significant genus member with respect to foodborne disease (Varnum and Evans, 1991). Traditional gastrointestinal symptoms, potentially mediated through the activity of a heat-stable enterotoxin, may develop into suppurative and autoimmune complications (Robins-Browne, 1997). The psychrotrophic status of Y. enterocolitica is potentially of great significance with regard to refrigerated MAP prepared produce. Y. enterocolitica occupies a broad range of ecosystems including the intestinal tract, birds, flies, fish and a variety of terrestrial and aquatic ecosystems. However, most environmental isolates lack virulence markers and are of doubtful significance for human or animal health (Delmas and Vidon, 1985). Isolation of Yersinia spp. from raw vegetables has been reported at rates ranging from 3.3% (Tassinari et al., 1994) to 46.1% (Delmas and Vidon, 1985), although specific isolation rates of pathogenic Y. enterocolitica strains are likely to be significantly lower. 12.2.7 Campylobacter jejuni Since their principal identification as human gastrointestinal pathogens in the 1970s (Butzler et al., 1973; Skirrow, 1977) members of the thermophilic campylobacters, e.g. C. jejuni, have emerged as major human gastrointestinal 238 Novel food packaging techniques

Reducing pathogen risks in MAP-prepared produce 239 pathogens(Ketley, 1997). Despite fastidious growth requirements, members of the genus survive at refrigeration temperatures for extended periods within nutrient limited environments. This property, combined with the low infective dose(robinson, 1981) and their microaerophilic nature, indicates the potential gnificance of the genus with respect to refrigerated MAP prepared produce Campylobacter are zoonotic pathogens, being primarily associated with the intestinal tracts of wild and domestic animals(Thomas et al, 1995)and are distributed throughout the environment through vehicles including birds, surface water and flies. Inappropriate food preparation and handling procedures may lead to the cross-contamination of fresh produce with Campylobacter from uncooked meats. and such errors could have resulted in the identification of MAP prepared vegetable products as sources of infection(Bean and Griffin, 1990, Altrkruse et al., 1994). A Canadian study of 296 fresh-cut MAP vegetable products detected no Campylobacter contamination(Odomeru et al, 1997) 12.2.8 Shigella species The genus Shigella is composed of four species, S. dysenteriae, S. sonnei, S boydii and S flexneri, all of which are pathogenic to humans at a low dose of infection. Fruits and vegetables may become contaminated with Shigella via fected food handlers or through the use of contaminated manure and irrigation water(FDA, 2001; Saddik et al, 1985). Several outbreaks of shigellosis have been attributed to contaminated produce(Freudland et al, 1987; see Table 12 and a 1986 outbreak of shigellosis was traced back to commercially distributed shredded packaged lettuce( Davis et al., 1988). Despite their mesophilic status Shigella can survive on lettuce stored at 5C for seven days(Davis et al., 1988) and on coleslaw at 4C for 16 days with numbers decreasing slightly during 12.2.9 Viral and protozoan pathogens The significance of viruses with respect to foodborne disease is clear with the inclusion of Norwalk virus, Hepatitis a virus and other viruses within the top ten causes of foodborne disease outbreaks in the USA(1983-1987, Cliver 1997). Outbreaks caused by hepatitis A virus, calicivirus and Norwalk-like viruses have been associated with the consumption of frozen raspberries and strawberries, melons, lettuce, watercress and diced tomatoes(Beuchat, 1996 Hedberg and Osterholm, 1993; Hutin et al., 1999: Lund and Snowdon, 2000 Rosenblum et al., 1990). Viruses can be transmitted by infected food handlers, through the fecal-oral route, and have been isolated from sewage and untreated water used for crop irrigation. Despite their significance, data regarding the effects of food preparation and storage conditions on the survival and infectivity of viruses is extremely limited, partly through the complexity of viral detection assays. Nonetheless, the potential of several viruses to survive on vegetables for periods exceeding their normal shelf-life has been identified(Badawy et al

pathogens (Ketley, 1997). Despite fastidious growth requirements, members of the genus survive at refrigeration temperatures for extended periods within nutrient limited environments. This property, combined with the low infective dose (Robinson, 1981) and their microaerophilic nature, indicates the potential significance of the genus with respect to refrigerated MAP prepared produce. Campylobacter are zoonotic pathogens, being primarily associated with the intestinal tracts of wild and domestic animals (Thomas et al., 1995) and are distributed throughout the environment through vehicles including birds, surface water and flies. Inappropriate food preparation and handling procedures may lead to the cross-contamination of fresh produce with Campylobacter from uncooked meats, and such errors could have resulted in the identification of MAP prepared vegetable products as sources of infection (Bean and Griffin, 1990; Altrkruse et al., 1994). A Canadian study of 296 fresh-cut MAP vegetable products detected no Campylobacter contamination (Odomeru et al., 1997). 12.2.8 Shigella species The genus Shigella is composed of four species, S. dysenteriae, S. sonnei, S. boydii and S. flexneri, all of which are pathogenic to humans at a low dose of infection. Fruits and vegetables may become contaminated with Shigella via infected food handlers or through the use of contaminated manure and irrigation water (FDA, 2001; Saddik et al., 1985). Several outbreaks of shigellosis have been attributed to contaminated produce (Freudland et al., 1987; see Table 12.1) and a 1986 outbreak of shigellosis was traced back to commercially distributed shredded packaged lettuce (Davis et al., 1988). Despite their mesophilic status, Shigella can survive on lettuce stored at 5ºC for seven days (Davis et al., 1988) and on coleslaw at 4ºC for 16 days with numbers decreasing slightly during storage (Rafii and Lundsford, 1997). 12.2.9 Viral and protozoan pathogens The significance of viruses with respect to foodborne disease is clear with the inclusion of Norwalk virus, Hepatitis A virus and ‘other viruses’ within the top ten causes of foodborne disease outbreaks in the USA (1983–1987; Cliver, 1997). Outbreaks caused by hepatitis A virus, calicivirus and Norwalk-like viruses have been associated with the consumption of frozen raspberries and strawberries, melons, lettuce, watercress and diced tomatoes (Beuchat, 1996; Hedberg and Osterholm, 1993; Hutin et al., 1999; Lund and Snowdon, 2000; Rosenblum et al., 1990). Viruses can be transmitted by infected food handlers, through the fecal-oral route, and have been isolated from sewage and untreated water used for crop irrigation. Despite their significance, data regarding the effects of food preparation and storage conditions on the survival and infectivity of viruses is extremely limited, partly through the complexity of viral detection assays. Nonetheless, the potential of several viruses to survive on vegetables for periods exceeding their normal shelf-life has been identified (Badawy et al., Reducing pathogen risks in MAP-prepared produce 239