O INSTITUT PASTEUR/ELSEVIER Bull. Inst. Pas teur Paris 1998 1998, 96, 151-164

REVIEW

Emerging and evolving microbial foodborne pathogens

J. Meng <]) ~*) and M.E Doyle (2)

~0 Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742 (USA)

~2) The Center for Food Safety & Quality Enhancement and Department of Food Science and Technology, University of Georgia, Griffin, Georgia 30223 (USA)

The epidemiology of foodborne diseases has changed rapidly in the last two decades. Emergence of newly recognized foodborne pathogens has significantly con- tributed to this change. Several pathogens have newly emerged as, or have the poten- tial to be, important foodborne pathogens, including Escherichia coil O157:H7 and other enterohaemorrhagic E. coll, Salmonella typhimurium Definitive Type 104, Cryptospori~ ium parvum, Cyclospora cayetanensis, Arcobacter butzleri and Helicobacter pylori. Oth- ers such as Campylobacter jejuni and Listeria monocytogenes have been recognized pathogens for many years but have only in the past two decades been determined to be predominantly foodborne. Although approaches such as hazard analysis critical control point (HACCP) programs will significantly improve safety of our food supply, changes in food processing, products, and practices, and human behaviour will influ- ence the emergence of foodbome pathogens into the next century.

I n t r o d u c t i o n

Foodborne diseases are reportedly an increas- ing public health problem worldwide. In the United States alone, foodborne infections each year cause millions of cases of illness and thou- sands of deaths; most infections go undiagnosed and unreported [1]. The epidemiology of food- borne diseases has changed in the last two decades partly because newly recognized patho- gens emerge and previously recognized patho- gens increase in occurrence or become asso- ciated with food or new food vehicles [2, 3]. Several pathogens have newly emerged as, or have the potential to be, important foodborne pathogens, including Escherichia coli O157:H7 and other enterohaemorrhagic E. coli, Salmo-

nella typhimurium Definitive Type 104, Crypto- sporidium parvum, Cyclospora cayetanensis, Arcobacter butzleri and Helicobacter pylori. Others, such as Campylobacterjejuni and Liste- ria monocytogenes, have been recognized patho- gens for many years but have only in the past two decades been determined to be predomi- nantly foodborne. In addition to acute gastroen- teritis, many of these foodborne pathogens may cause severe sequelae or disabi l i ty . E. coli O157:H7, for example, is a leading cause of haemolytic uraemic syndrome in children in the United States. Salmonella causes not only inva- sive i n f ec t i ons but also r eac t ive ar thr i t i s . Recently, Campylobacter has been reported to be one of the most common causes of Guillain- Barr6 syndrome (GBS). This article reviews dis-

Submitted April 1, 1998, accepted May 6, 1998.

(*) Corresponding author.

152 J. M E N G A N D M . P . D O Y L E

eases caused by these emerging or evolving microbial pathogens and their significance as foodborne pathogens.

E. coil O157:H7 and other enterohaemorrhagie E. coli

E. coli O157:H7 was first recognized as a human pathogen in 1982 when two outbreaks of haemorrhagic colitis were associated with con- sumption of undercooked ground beef that had been contaminated with this organism [4]. Since then, E. coli O157:H7, O111:NM (non-motile) and several other serotypes of Shiga toxin-pro- ducing E. coil (STEC) have caused a large num- ber of foodborue outbreaks of haemorrhagic col- itis and haemolytic uraemic syndrome (HUS) worldwide [5, 6]. Large outbreaks of E. coli O157:H7 infections include a 1992-93 outbreak that affected more than 700 individuals in the western United States [7] and 1996 outbreaks in Japan in which more than 8,000 cases were reported [8].

The term "en te rohaemorrhag ic E. coli'" (EHEC) was originally given to strains that cause haemorrhagic colitis and HUS, express Shiga toxins (verotoxins), cause attaching and effacing lesions on epithelial cells, and possess a ca. 60- MDa plasmid [5]. The ability to produce Shiga toxin was acquired from a bacteriophage, pre- sumably directly or indirectly from Shigella. Thus, EHEC denotes a subset of STEC and includes a clinical connotation that is not implied with STEC. Whereas not all STEC strains are believed to be human pathogens, all EHEC strains by the above definition are considered to be pathogens. The initial symptoms of haemor- rhagic colitis caused by E. coli O157:H7 gener- ally occur 1-2 days after eating contaminated

food. Symptoms begin with mild, non-bloody diarrhoea that may be followed by a period of "crampy" abdominal pain and short-lived fever [9]. The initial diarrhoea increases in intensity during the next 24-48 h to a 4-10-day phase of overtly bloody diarrhoea accompanied by severe abdominal pain and moderate dehydration. Life- threatening complications may occur in patients suffering from haemorrhagic colitis; HUS is the most common. HUS occurs most often in chil- dren under the age of 10. Approximately half of HUS patients require dialysis, and the mortality rate is 3-5 %. A second complication is throm- botic thrombocytopenic purpura. This condition resembles HUS, except that it generally causes less renal damage, has significant neurological involvement, e.g., central nervous system deteri- oration, seizures and strokes, and occurs primar- ily in adults.

Cattle [10, 11] and other ruminants [12, 13] are considered to be the major reservoirs of STEC including E. coil O157:H7, although STEC have also been isolated from other animals ,such as dogs, horses, swine and cats [14, 15]. A study on STEC infection on dairy farms in Can- ada revealed that 36 % of cows and 57 % of calves were STEC-positive in all of the 80 herds tested 1116]. On four farms, the same STEC serotypes were identified in cattle and humans. Seven ani- mals (0.45 %) on four farms (5 %) were positive for E. coli O157:H7. Results of two major sur- veys in the United States indicated that 31 (3.2 %) of 965 dairy calves and 191 (1.6%) of 11,881 feedlot cattle were positive for E. coil O157:H7 [11, 17]. An additional 0.4% of feedlot cattle were positive for E. coli O157:NM. E. coli O157:H7 levels in calf faeces ranged from <102 CFU/g to 105 CFU/g [11]. Prevalence of E. coil O157:H7 in sheep is higher. A six-month study of healthy ewes revealed that faecal shed-

ACSSuT =

CDC CFU = EHEC = ELISA = GBS =

ampicillin, chloramphenicol, streptomycin, suiphonamides and tetracyclines (resistance pattem). Centers for Disease Control and Prevention. colony-forming unit(s). enterohaemorrhagic E. coli. enzyme-linked immunosorbent assay. Guillain-Barr6 syndrome.

ttACCP =

HUS = LPS = PCR = R-type = STEC = Tm =

hazard analysis critical control point (programs). haemolytic uraemic syndrome. lipopolysaccharide. polymerase chain reaction. resistance pattern. Shiga toxin-producing E. coli. trimethoprim.

EMERGING AND EVOLVING MICROBIAL FOODBORNE PATHOGENS 153

ding of the pathogen was transient and seasonal, with 31% of sheep E. coli O157:H7-positive in June, 5.7 % positive in August and none in November [12]. Animals carrying E. coli O157:H7 appear healthy.

Contamination of meat with STEC during slaughter is a principal route by which these pathogens enter the food supply. In view of the high rates of faecal carriage of STEC by cattle and other animals, it is not surprising that meats are contaminated with STEC. Several studies have determined that ground beef has high con- tamination rates of STEC in Canada (15 to 40%) [18], the United States (23 to 25 %) [19, 20], the United Kingdom (17 %) [21] and the Netherlands (16.1%) [22]. One study determined STEC in 63 % of veal and 48 % of lamb samples from retail stores in Seattle, Washington, USA, although sample size (8 veal and 21 lamb sam- pies) was small [19]. Using Shiga toxin gene probes, the same study also detected STEC in 18 % of pork, 12 % of chicken, 7 % of turkey, 10% of fish and 5% of shellfish samples. In con- trast, E. coli O157:H7 was isolated from only 0 to 2 % of the samples assayed in the Canadian stud- ies, and no E. coli O157:H7 was detected in the other studies described above. In an earlier study, Doyle and Schoeni [23] isolated E. coli O157:H7 from 6 (3.7%) of 164 beef samples, 4 (1.5%) of 264 pork samples, 4 (1.5%) of 263 poultry sam- pies, and 4 (2 %) of 200 lamb samples obtained from retail stores in Wisconsin, USA and Alberta, Canada.

Many outbreaks of E. coli O157:H7 infection have been associated with the consumption of undercooked ground beef [6]. Other foods have also been associated with E. coli O157:H7 out- breaks worldwide, including roast beef, venison jerky, salami, raw milk, pasteurized milk, yogurt, lettuce, unpasteurized apple cider/juice, canta- loupe, radish sprouts and alfalfa sprouts; an out- break associated with handling potatoes was reported in the United Kingdom [24]. It is note- worthy that certain foods such as apple cider and dry-cured salami that previously were considered safe and ready to eat because of their high acidity, and are usually not heated before consumption, have been vehicles of outbreaks. The pathogen

has been shown experimentally to survive for several weeks to months in a variety of acidic foods, including mayonnaise, sausages, apple cider, and Cheddar cheese [24]. Sprouts were recently added to the spectrum of vehicle foods transmitting E. coli O157:H7 infection. Radish sprouts were implicated as the vehicle of the 1996 outbreaks in Japan. More recently, two out- breaks in Michigan and Virginia in the United States were associated with consumption of alfalfa sprouts [25]. The seeds used to grow the sprouts were from the same distributor, and the contamination most likely occurred in alfalfa seeds rather than during the sprouting process. Reports of person-to-person and waterborne (par- ticularly recreational lake water) transmission have been increasing (CDC, 1997, personal com- munication).

Most outbreaks of EHEC have been associated with serotype O157:H7 in North America and Europe [5, 26], but several were caused by EHEC belonging to serotypes other than O157:H7. Out- breaks caused by E. coli O111:NM and 026 have been n:ported in Italy [27]. E. coli O103:H2 was isolated from patients suffering from HUS in France [28]. In Japan, EHEC serotypes O? :HI9, O l l l : N M , O145:NM and O118:H2 were the cause of several outbreaks [8]. Studies in Austra- lia have revealed that serotype O157:H7 was uncommon but other less well-recognized sero- types such as Ol l l :NM, O6:H31, O98:NM and O48:H7 were the causative agents of haemor- rhagic colitis and HUS [29]. However, in contrast to E. coli O157:H7 outbreaks in which food is frequently identified as a vehicle, the mode of transmission of most outbreaks caused by non- O157 EHEC has not been identified [18]. Only two recent outbreaks of non-O157 EHEC infec- tion have been clearly associated with foods; E. coli O111:NM in fermented sausage caused an outbreak in southern Australia [30], and milk contaminated with O 104:H21 was responsible for an outbreak in Munt,u~,,, USA [31]. Twenty-three cases of HUS among children under 10 years of age were reported in the Australian outbreak. E. coli Ol l l :HM was isolated from stool specimens as well as sausage samples obtained from the homes of palients, the same manufacturer and retail stores. In contrast, E. coli O104:H21 was not iso-

154 J. MENG AND M.P. DOYLE

lated from milk in the Montana outbreak, and HUS was not reported among the 18 patients. The potential for foodborne transmission of EHEC other than E. coli O157:H7 needs to be more fully elucidated.

S. typhimurium DT104

The incidence of reported cases of salmonello- sis has increased significantly in many areas of the wodd during the past two decades, particu- larly since the mid 1980s. In England and Wales, Salmonella isolations reported from human infec- tions doubled from 10,251 in 1981 to 22,627 in 1991 [32]. Recent major changes in the epidemi- ology of salmonellosis have been the emergence and increase of Salmonella enteritidis in industri- alized countries and S. typhimurium definitive phage type 104 (DT104) in the United Kingdom, other European countr ies such as Germany, France, Austria and Denmark, and in the United States [33]. Of particular importance has been the epidemic spread of antibiotic-resistant DT104 that was first identified in humans in England and Wales in 1984. The organism has an antimicro- bial resistance pattern (R-type) to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT). The number of cases of DT104 infection increased from 259 in 1990 to 4,006 in 1996 in England and Wales [34]. The prevalence of DT104 infection in the United States is less clear. In a retrospective study, Bes- ser et al. [35] reported that S. typhimurium with R-type ACSSuT was absent in 44 cattle isolates obtained before 1986 in the Pacific Northwest region, but accounted for 13 % of 83 isolates obtained between 1986 and 1991 and for 64% of 51 isolates obtained since 1992. Sixty of these isolates were phage typed and 57 were DT104. A study by the U.S. Centers for Disease Control and Prevention (CDC) revealed that S. typhimurium with the same antibiotic resistance pattern (R- type ACSSuT) was present in 90 (32 %) of the 282 human isolates tested in 1996 [36]. This pat- tern also was present in 272 (28 %) o f 976 S. typhimurium isolates identified during a 1995 national survey, compared with 7% in 1990. Among thirty isolates of S. typhimurium R-type

ACSSuT obtained from 10 states in 1995, 25 (83 %) were identified as DT104.

The emergence of multidrug-resistant strains reduces the therapeutic options in cases of inva- sive infections and could have serious public health implications. Unfortunately, the spectrum of antibiotic resistance in S. typhimurium is still increasing. In addition to the common R-type ACSSuT, resistance of DT104 isolates to other antibiotics such as trimethoprim (Tin) and cipro- floxacin has been reported [33]. DT104 isolates with R-type ACCSSuTTm increased from 12% in 1994 to 27% in 1995 in England and Wales. DT104 isolates with additional resistance to cip- rofloxacin increased from 1% to 6% in the same period. Because ciprofloxacin is now the drug of choice for treatment of invasive salmonellosis in humans, the appearance of resistance to this anti- microbial in strains of DT104 that are already resistant to other antibiotics is of particular con- cern. Furthermore, DT104 is unusual in that the resistance genes have become chromosomally integrated and can be retained in the absence of selective pressure. Therefore, it is unlikely that withdrawal of antibiotics would have any signifi- cant effect on resistance.

Human infections with non-typhoid salmonel- lae c o m m o n l y result in enterocol i t i s , which appears 8 to 72 h after contact with the invasive pathogen [37]. The clinical condition is generally self-limiting, and remission of the characteristic non-bloody diarrhoeal stools and abdominal pain usually occurs within 5 days of onset of symp- toms. Invasiveness of DT104 in humans does not appear to differ from other salmonellae; however, an increase in occurrence of severe illness has been reported, with 36% of 105 patients in one case-control study requiring hospitalization [32]. In a 1993 epidemiologic study, 34 of 83 DT104 cases required hospitalization and 10 died [38]. A national case-control study in the United States also showed that infection with DT104 R-type ACSSuT was associated with higher hospitaliza- tion and fatality rates than other Salmonella sero- types were. In addition to the pathogen's acute health effects that often last for a week, salmonel- losis may also lead to serious chronic complica- tions such as aseptic reactive arthritis, Reiter's

EMERGING AND EVOLVING MICROBIAL FOODBORNE PATHOGENS 155

syndrome and ankylosing spondylitis [37]. The very young and the elderly are most susceptible to serious complications.

In contrast to S. enteritidis, which is associated primarily with poultry and eggs, epidemiologic evidence indicates that DT104 is widely distrib- uted in a variety of different food animals includ- ing cattle, sheep, pigs, goats, chickens and tur- keys as well as domestic pets. The pathogen causes severe diarrhoea in cattle, and a mortality rate of 50-60% has been reported for animals c l inical ly affected in some outbreaks in the Uni ted Kingdom [33]. A major i ty of recent bovine salmonellosis cases investigated in Eng- land were caused by DT104, and 15 of 16 new outbreaks of bovine salrnonellosis in Scotland in March 1996, were caused by DT104 [39]. Long- term carriage (up to 18 months following an out- break) has been observed in all species, particu- lar ly in cats [40] and ca t t le [41]. Of 1 10 Salmonella spp. isolated from sick house cats in England in 1991-1994, 40 were S. typhimurium DT104 that were resistant to 5 antibiotics. Con- tact with sick animals has been identified as a sig- nificant risk factor. A number of farm families appear to have acquired DT104 infections while caring for sick farm animals. Household pets may also be a source of infection.

Foodborne transmission of DT104 infection has been well documented. Investigations of 46 outbreaks of DT104 infections in the UK from 1992 to 1996 revealed that 78% of the outbreaks were linked to foodborne transmission and that 15 % were attributed to contact with infected ani- mals [33]. Suspected food vehicles included roast beef, ham, pork sausage, salami sticks, "cooked meats" and chicken, as well as pas teur ized/ unpasteurized milk, and chocolate milk [42, 43]. There have been five outbreaks of human infec- tion identified in the United States (two in Cali- fornia, one each in Washington, Nebraska and Vermont) [33]. Four of these outbreaks involved consumption of unpasteurized dairy products or contact with dairy cattle. The pathogen also has been isolated from a wide range of food products. Analysis of 786 samples of fresh and frozen sau- sages in England in 1994 demonstrated that 17% were contaminated with Salmonella spp. includ-

ing DT104 [33]. This indicates that these bacteria are commonly present in some types of meats and pose a significant risk if such foods are not cooked and handled properly.

L. monocytogenes

This organism has been recognized as a cause of human illness for more than 60 years; how- ever, the first confirmed foodborne transmission of listeriosis was not demonstrated until 1981 [44]. Since then, epidemiologic investigations have repeatedly revealed that contaminated food is a primary vehicle of transmission of listerio- sis. Listeriosis is an atypical foodborne disease of major public health concern because of the severity and non-enteric nature of the disease (meningitis, septicaemia and abortion), and a high case-fatality rate (ca. 20 to 30 % of cases) [44]. Although listeriosis can occur in otherwise healthy adults and children, immunocomprom- ised individuals inc lud ing the i m m u n o s u p - pressed, elderly, pregnant and those suffering a range of underlying diseases are primarily at risk [44].

L. monocytogenes differs in many respects f rom most other foodborne pathogens . It is widely distributed, if not ubiquitous, in soil, sew- age, fresh water sediments and effluents, and is frequently carded in the intestinal tract of animals and humans [45]. Its occu r rence in hea l thy humans and animals has been documented since 1965. R e c e n t s tudies on the p r eva l ence o f L. monocytogenes in humans indicated that 2 to 6 % of individuals were positive. A more frequent faecal carrier state (10 - 50%) has been docu- mented in animals, including cattle, poultry and swine. L. monocytogenes has been isolated from a variety of food products, including fresh vegeta- bles (11%), raw meats (13%), raw milk (3-4%), dairy products (3 %), eggs and seafood products [46]. The bacterium grows well at refrigeration temperature and in minimal nutrients, and is able to survive and even multiply on plants, and in soil and water. The w i desp read d i s t r ibu t ion of L. monocytogenes enables its frequent contamina- tion of food products during various phases of

156 J. MENG AND M.P. DOYLE

production, processing and manufacture, and dis- tribution. These characteristics have made the pathogen a major nemesis to the food industry. Foods associated with outbreaks have largely been refrigerated, processed and ready-to-eat foods, and consumed without prior cooking or reheating. Examples include coleslaw, pasteur- ized milk, soft cheese, patt, pork tongue in jelly, shrimp and smoked mussels [45]. Some foods appear to be of greater risk than others, particu- larly those that support the growth of L. monocy- togenes and are ready-to-eat and stored at refrig- eration temperature for a long period.

The incidence of listeriosis is relatively low, considering the widespread presence of L. mono- cytogenes in the environment, households and foods; most of the human population frequently ingests listeriae without ill effects. Hence, some unique host factors apparently predispose certain individuals to listeriosis, with most persons apparently resistant to listeriae infection [44]. In addition to host factors and exposure to specific foods, it is likely that microbial characteristics also are important risk factors for disease. Poten- tial virulence factors as well as infectious dose could affect the occurrence and course of infec- tion. Human strains associated with outbreaks as well as 45 to 70% of strains responsible for spo- radic cases belong to serovar 4b, to two major pro- files of isoenzymes and to one major ribovar [47]. In contrast, serogroup 1/2 strains account for most food and environmental isolates.

Since the first confirmed foodborne outbreak in 1981, several outbreaks of foodborne listeriosis have been reported in North America and Europe. More recently in France, two major outbreaks occurred in 1992 and 1993, the first involving 279 cases with 85 deaths [44] and the 1993 out- break involving 38 cases [48]. These outbreaks were each linked to processed, ready-to-eat pork products contaminated with L. monocytogenes. An outbreak associated with raw milk soft cheese was also reported in France in 1995 [44]. Out- breaks of a milder form of listeriosis have been documented recently. An outbreak of listeriosis in Italy in June 1994, involving 39 cases [49], had 70% of the patients with gastroenteritis and 30% with a flu-like illness. Rice salad was implicated

as the vehicle, and L. monocytogenes was isolated from three leftover foods, the kitchen freezer and blender. Isolates from the patients, the foods and the freezer were indistinguishable and belonged to serotype 1/2. A recent outbreak occurred at a dairy cattle convention in Illinois in July 1994, with 52 of 64 otherwise heal thy individuals developing mild gastrointestinal illness [50]. Chocolate milk was identified both epidemiolog- ically and by microbiologic culture as the vehicle of infection.

Studies by the CDC reported a reduction in the incidence of listeriosis in the United States [51]. Invas ive d i sease due to L. monocy togenes decreased by 44% from 1989 through 1993. Lis- teriosis-related deaths decreased by 48% during the same period. The number of estimated cases was 1,092 annually, with 248 deaths, compared with an estimated 1,965 cases with 481 deaths in the mid to late 1980s. Efforts by the U.S. food industry and governmental regulatory agencies to reduce contamination by listeriae in foods may be responsible for this reduction in cases of listerio- sis. A zero tolerance policy for L. monocytogenes in all ready-to-eat food products was established in 1986 by United States regulatory agencies (Food and Drug Administration and U.S. Depart- ment of Agriculture) in responding to concems about foodbome listeriosis. The policy requires ready-to-eat foods to be negative for L. monocy- togenes in two 25-gram samples of the food prod- uct. This policy was established based on mini- mal information about foodborne Listeria at that time. With a more complete understanding of the occurrence, transmission and control of L. mono- cytogenes as a foodborne pathogen, many micro- biologists now believe that it is time to reevaluate and establish tolerances for L. rnonocytogenes in low-risk foods such as acidic and frozen foods that do not allow growth of the bacterium.

C. jejuni

C. jejuni is one of 20 species and subspecies within the genus Campylobacter, family Campy- lobacteraceae, which also includes four species in the genus Arcobacter. The organism was not

EMERGING AND EVOLVING MICROBIAL FOODBORNE PATHOGENS 157

recognized as a cause of human illness until the late 1970s and is now considered the leading cause of foodborne bacterial infection [3]. C. jejuni and C. coli are the most common Campylobacter spe- cies associated with diarrhoeal illness and are clin- ically indistinguishable. The relative ratio of C. jejuni to C. coli is not known because most labor- atories do not routinely distinguish these organ- isms. In the United States, an estimate of approxi- mately 5 to 10% of cases reported as due to C. jejuni are due to C. coil [52]. Campylobacter usu- ally causes an acute, self-limiting enterocolitis last- ing up to a week, but relapses may occur in 5 to 10 % of untreated patients [52]. Symptoms and signs usually include fever, abdominal pain and diar- rhoea. Two types of diarrhoea are observed with campylobacter enteritis: inflammatory diarrhoea, with fever and slimy, often bloody stools contain- ing leukocytes; and non-inflammatory diarrhoea, with watery stools and the absence of leukocytes and blood. Bacteraemia and reactive arthritis are rare complications. C. jejuni infection has also been the most frequently identified cause of GBS, which is defined clinically as a peripheral neurop- athy causing limb weakness that progresses for up to 4 weeks before reaching a plateau [53]. Deaths at tr ibutable to C. jejuni infect ion have been reported but rarely occur.

Little is known about the mechanism by which C. jejuni causes human disease. By analogy to other enteropathogens, and considering the typi- cal motility of C. jejuni, four major virulence properties have been identified: motility, adher- ence, invasion and toxin production [54]. Motility is not only required for the bacteria to reach the attachment sites but is also required for their pen- etration into intestinal cells, although the exact role of f lagella in this process has not been defined. Adherence of bacteria to the epithelial surface is important for colonization and may increase the local concentration of secreted bacte- rial products . However , specif ic adhesins of C. jejuni have not been identified. Fimbrial struc- tures on the cell surface were associated with col- onization in rabbit ileal loops. Invasion has been shown and putative factors required for invasion have been identified. However, invasion levels as detected in vitro are normally low; less than 1% of the applied bacteria invade a monolayer of cell

culture, and efficient intracellular killing of bacte- ria takes place [55]. Several toxins including enterotoxins and cytotoxins produced by C. jejuni have been reported [56]. However, their mecha- nisms of action and their importance in disease remain unclear.

C. jejuni infection can lead to autoimmune sequelae, GBS, which may arise as a result of the production of antibodies to the lipopolysaccha- ride (LPS) of C. jejuni that, due to molecular mimicry, cross-react with gangliosides or other structures present in peripheral nerves. Studies revealed that strains belonging to serotype O:19 as well as some other serotypes (0:4, O:1) of C. jejuni have core oligosaccharide LPS struc- tures that mimic ganglioside motor neurons [57].

C. jejuni is associated with warm-blooded ani- mals but does not survive well outside of the host at room temperature or above. The organism is part of the natural intestinal flora of a wide variety of wild and domestic animals, with poul- try being prominent [58]. Its prevalence in faecal samples often ranges from 30 to 100% [52, 59]. It has been isolated from many uncooked foods of animal origin at retail stores. U.S. Department of Agriculture baseline studies of ready-to-cook poul~¢ following processing at slaughter plants revealed that 88 % of broilers were positive for C. jejuni [59]. Fortunately, C. jejuni is susceptible to a w~riety of environmental conditions. It does not grow or survive well in food, and is relatively fragile and readily killed by heat treatments used to cook foods. Hence, food-associated illness often results from eating foods of animal origin that are raw or inadequately cooked, or that are recontaminated after cooking by contact with C. jejuni-contaminated raw materials. However, the infectious dose of C. jejuni can be quite low, with a few hundred cells causing illness [52]. The organism is among the most common causes of sporadic bacterial enteritis, and this is in contrast to the relatively low occurrence of outbreaks caused by C. jejuni [60]. The source of infection in indiLvidual cases usually is not identified. Nev- erthelless, spo rad ic cases and o u t b r e a k s of C. jejuni infection are likely to be caused by con- sumption or contact with contaminated poultry meat, milk, or water, or by contact with animals

158 J. MENG AND M.P. DOYLE

or birds [61, 62]. Results of some epidemiologic studies have provided estimates that approxi- mately 50 % of sporadic cases of campylobacter enteritis are associated with handling or eating poultry [59]. A recent outbreak in the United States was associated with vegetable salad cross- contaminated by raw chicken in a restaurant [63]. Fourteen persons became ill 1 to 5 days after eat- ing the contaminated salad, and two were hospi- talized.

The apparent high degree of virulence of the organism, as indicated by its relatively low infec- tious dose, coupled with its widespread preva- lence in foods of animal origin, is important in explaining why this sensitive bacterium is a lead- ing cause of gastroenteritis. A CDC report result- ing from data derived from the Foodborne Dis- ease Active Surveillance Network (FoodNet) revealed that campylobacter enteritis was the most common of the enteric infections, with an incidence rate of 25 per 100,000 population, fol- lowed by salmonellosis (16 per 100,000), shigel- losis (9 per 100,000) and E. coli O157:H7 infec- tion (3 per 100,000) [64]. C. jejuni has also emerged as the leading cause of infective diar- rhoea in humans in England and Wales, but few outbreaks have been reported [60].

A. butzleri and H. pylori

A. butzleri and H. pylori formerly belonged to the genus Campylobacter. However, in 1991, those bacteria that were known as aerotolerant Campylobacter or Carnpylobacter-like organisms were clarified as Arcobacter. Vandamme pro- posed that the rRNA superfamily VI of the Pro- teobacteria include Arcobacter, Campylobacter and Helicobacter [65]. Arcobacter spp. primarily differ from Campylobacter spp. in their ability to grow under aerobic conditions at 15°C. These spiral organisms have been associated with abor- tions and enteritis in animals; only enteritis has been observed in humans. Arcobacters have been isolated from domestic animals, humans, poultry, ground pork and drinking water [66, 67]. Two species o f Arcobacter (A. cryaerophilus and A. butzleri) have been associated with human

disease. Most of the isolates from humans belong to the species A. butzleri [68].

The evidence that Arcobacter spp. are patho- genic is based on their more frequent recovery from aborted pig litters and from infertile sows with vaginal discharge than from healthy animals. A. butzleri has been cultured from humans with enteritis who were otherwise healthy and from patients suffering from diarrhoea with chronic underlying disease. There is very limited infor- mation about the clinical significance, pathoge- nicity and epidemiology of A. butzleri. One study revealed that more than 50% of patients with A. butzleri infection suffered from persistent diarrhoea accompanied by abdominal pain, nau- sea and fever [68]. However, ten Italian school children involved in a 1983 outbreak had recur- rent abdominal cramps without diarrhoea [69].

T ransmiss ion of A. butzleri may involve 1) drinking contaminated water associated with travel, 2) consumption of contaminated food, or 3) person-to-person contact [70]. Although food has not been identified to be associated with Arcobacter infection, the fact that the organism causes diseases in domestic animals and has been isolated from meat products and water increases the likelihood of Arcobacter as a potential food- borne pathogen.

Since its first identification in 1982, H. pylori, formerly known as Campylobacter pylori, has been implicated as the aetiological agent of gas- tritis and as a major contributing factor in the development of peptic gastroduodenal ulcers [71]. H. pylori also has been associated with gas- tric cancer. H. pylori infection occurs worldwide and is frequently asymptomatic, particularly in children and young adults [71]. In developing countries, the infection occurs more frequently at younger ages and reaches prevalences of 70 % to over 90% in some regions. In developed coun- tries, the prevalence of infection is lower, ranging from 25 to 50%. The data from developed coun- tries also sugges t that mos t i n f ec t i ons are acquired in childhood. H. pylori infection, once acquired, persists for years, decades, or possibly for life [72]. Spontaneous eradication of infection with gastric healing may occur. Eradication of H. pylori usually results in a healing, or at least a

EMERGTNG AND EVOLVING MICROBIAL FOODBORNE PATHOGENS 159

pronounced improvement, of gastritis in adults and children. Chronic superficial gastrit is caused by H. pylori may be either symptomatic or asymptomatic. Early in ~he course of infec- tion there may be changes in acid secretion. Later in the course, infection may be associated with non-ulcer dyspepsia, peptic ulcer disease, type B atrophic gastritis or gastric carcinoma [71]. The progression of H. pylori infection from chronic superficial gastritis to one of these syndromes may require cofactors, including genetic predisposition, smoking, alcohol and diet. Differences in the virulence properties of H. pylori strains may also have an influence on disease progression.

The human originally was thought to be the only natural host of H. pylori. However, the organism has also been isolated from non-human primates [73] and very recently from cats [74], suggesting that the organism may be a zoonotic pathogen, with transmission occurring from ani- mals to humans. H. pylori is quite fragile and does not thrive well outside of its host. Although the pathogen has been detected in drinking water and vegetables by using PCR and ELISA [75], currently available techniques have had limited success in isolating viable H. pylori from poten- tial environmental sources and foods. Various evidence supports the hypothesis of waterborne transmission. H. pylori could survive for more than one year in coccoid forms in a river water microcosm and remain viable and culaarable for more than 10 days in fiver water at 4°C [75]. The significance of H. pylori as a foodborne pathogen requires further elucidation [70].

C. parvum and C. cayetanensis

Intestinal protozoa C. parvum and C. cayeta- nensis (previously known as cyanobacterium or blue-green algae-like organism) are not new organisms, both having been first described many decades ago [76]. However, only recently were they implicated as important water- and food- borne pathogens, following several large out- breaks of human illness. Although C. parvurn can

infect healthy individuals, the AIDS epidemic has played a major role in the recognition and clinical management of this recently emerged water- and foodborne pathogen. C. parvum and C. cayeta- nensis share many common characteristics in biology, pathogenesis, epidemiology, clinical manifestations and diagnosis [76]. Cyclosporasis is treatable with trimethoprim-sulphamethoxazole; however, not cryptosporidiosis. Many animal spe- cies have infections with C. parvum and Cyclo- spora. However, the species of intestinal Cyclo- spora found in humans are different from those in animals. C. cayetanensis is the only member of the genus known to infect humans. In contrast, the same species C. parvum that causes diseases in humans is also an important cause of disease in cattle, goats and other farm animals. In a nation- wide survey conducted by the U.S. Department of Agriculture, C. parvum oocysts were found in calves from 59.1% of 1,103 farms and in 22.4% of the 7,369 tested calves [77]. Recent studies revealed that Cryptosporidium oocysts were present in 65 to 97 % of surface waters (rivers, lakes, etc.) tested throughout the United States [78]. There is no information available on the prevalence of C. cayetanensis in animals and in the environment.

The pathogenesis of C. parvum and C. cayeta- nensis is not clear. Possibly, they invade entero- cytes, primarily in the small intestine, resulting in substantial alterations in intestinal structure and function [76]. Symptoms of cryptosporidiosis and cyclosporiasis are similar in normal hosts, includ- ing watery diarrhoea (often 4 weeks for Cyclo- spora), nausea, anorexia, abdominal cramping, fever and weight loss. Persons with immunodefi- ciency are predisposed to more frequent, pro- longed and life-threatening diarrhoea.

Numerous, well-documented outbreaks of cryptosporidiosis have occurred. Most of these were waterbome outbreaks that involved subtle problems in the flocculation and/or filtration process. A 1993 outbreak in Milwaukee, Wis- consin, was the largest in U.S. history and affected an estimated 403,000 persons, with a 52% attack rate among those served by the South Milwaukee water plant [79]. Other outbreaks involved public swimming pools and wading

160 J. MENG AND M.P. DOYLE

pools [80, 81]. This further confirmed the ability of Cryptosporidium to cause infect ion even when ingested in relatively small amounts of fully chlorinated water. Several foodborne out- breaks also have been documented. Implicated food vehicles include apple cider [82], chicken salad [83] and pasteurized milk [43]. An out- break associated with fresh-pressed apple cider in Maine, USA affected 54% of those ingesting the contaminated cider [82]. Cryptosporidium oocysts were found in the apple cider, cider press, as well as in a ca l f on the farm from which the apples were obtained.

Before 1996, most reported cases of cyclospo- riasis afflicted international travelers. Since then, there have been several large outbreaks involving this parasite in North America. In a 1996 out- break, a total of 1,465 cases of cyclosporiasis were reported in 20 states and the District of Columbia in the United States, and 2 provinces in Canada [84]. Raspberries imported from Guate- mala were implicated as the vehicle of transmis- sion. The parasite, however, was never detected in the fruit, due in part to the lack of effective detec- tion methods. It is not known how the raspberries became contaminated. One hypothesis is that water used to dissolve and spray pesticides was contaminated with the parasite. Several outbreaks and sporadic cases, involving more than 1,600 cases, also were reported in 1997 [85, 86]. In addition to fresh raspberries from Guatemala, mesclun (a mixture of various types of baby leaves of lettuce) [85] and basil used in a pasta sauce [86] were associated with outbreaks. How- ever, the specific sources of contamination of the implicated mesclun and basil were not deter- mined.

Cryptosporidium oocysts have been detected in fresh vegetables, raw milk, sausage and tripe [87, 88]. Faeces from infected persons or animals are the likely source of contamination of foods. Although transmission of C. cayetanensis is less clear, its involvement in several foodborne out- breaks suggests the organism could be transmit- ted in much the same way as C. parvum. Uncooked contaminated foods may be vectors of transmission of oocysts to consumers. Cyclospora sporosites require a holding time (1 week) out-

side the host to be activated in order to be infec- tive. Hence, the food handler theory needs eluci- dation.

Concluding comments

Several newly recognized foodborne patho- gens have contributed greatly to the occurrence of foodborne diseases during the last two decades. Along with emerging/evolving pathogens, new food vehicles of transmission also have been implicated in recent years. Foods previously thought to be safe are now considered potentially hazardous. Many foodborne pathogens have an animal reservoi r f rom which they spread to humans, but several do not cause illness in the infected host animal. Hence there is often no obvious indicat ion that the source animal is infected. In addition, the foods contaminated by these pathogens frequently look, smell and taste normal. Control and prevention of foodborne dis- eases can be difficult, as each segment in the pro- duction, preparation and delivery of food must do its part to reduce or eliminate foodborne patho- gens. Approaches such as hazard analysis critical control point (HACCP) programs can play an important role in reducing foodborne infections. However , solut ions which would comple te ly eliminate such pathogens from food are complex and not readily available. Changes in food pro- cessing in new product formulations, in food han- dling practices, and people's attitudes toward eat- ing raw or undercooked foods of animal origin will continue to facilitate the emergence of food- borne pathogens into the next century. As ade- quate cooking and irradiation are the most effec- t ive means to inac t iva te mos t f o o d b o r n e pathogens in food, it is unlikely that consumers will be able to safely enjoy certain raw (fresh) or undercooked foods. Innovative approaches for pathogen control, from reducing contamination to treating foods to kill foodborne pathogens and retain freshness and flavours, are critically needed.

Key-words: Food, Microflora; Epidemiology, Emerging pathogen, Evolution, Public health; Review.

E M E R G I N G A N D E V O L V I N G M I C R O B I A L F O O D B O R N E P A T H O G E N S 161

l~volution de l'~pid~miologie des microorganismes pathog~nes ~mergents

v~hicul~s par les aliments

L'EpidEmiologie des maladies transmises par les microorganismes portEs par les aliments a connu une Evolution rapide au cours des deux derniEres decades . L ' E m e r g e n c e de n o u v e a u x agents pathog~nes d'origine alimentaire a contribuE grande- ment a c e changement. Plusieurs agents pathogbnes ont rEcemment Emerge comme Etant dEj~ ou poten- tiellement puissants et vEhiculEs par les aliments, ce qui inclut E s c h e r i c h i a col i O157:H7 et d 'autres E. coli entErohEmorragiques, Salmonella typhimurium DT104, Crytosporidum parvum, Cyclospora cayeta- nensis, Arcobac ter butzlerii et Helicobacter pylori. D'autres agents reconnus comme pathogEnes depuis longtemps, tels C a m p y l o b a c t e r j e j u n i et Lis ter ia monocytogenes , ont Et6 caractErisEs, seulement dans les 20 derni~res annEes, comme agents vEhiculEs essent ie l lement par les al iments. Bien que les approches comme le programme HACCP (hazard analysis critical control point) soient prEvues pour amEliorer la sEcurit6 de nos approvisionnements ali- mentaires, les modif icat ions dans le trai tement industriel des aliments, des produits et des proto- coles, et le cornportement humain auront une influ- ence dans l 'Emergence d 'agents pathog~nes <<ali- mentaires >>.

Mots-clds: Aliment, Microflore; I~pidEmiologie, Agents pathog~nes Emergents, [~volution, SantE publique ; Revue.

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