Viruses and Some Other Proven and Suspected Foodborne Biohazards 733 The bacteria most often associated with this syndrome are Morganella spp., especially M morganii, of which all strains appear to produce histamine at levels >5,000 ppm Among other bacteria shown to produce histidine decarboxylase are Raoulella planticola and R ornithinolytica,55 and Hafnia alvei, Citrobacter freundii, Clostridium perfringens, Enterobacter aerogenes, Vibrio alginolyicus and Proteus spp A Morganella morganii isolate from temperature-abused albacore produced 5,253 ppm histamine in a tuna fish infusion medium at 25◦ C, and 2,769 ppm at 15◦ C.59 Neither growth nor histamine production occurred at 4◦ C P phosphoreum produces histamine at temperatures at and below 10◦ C From room-temperature spoiled skipjack tuna, 31% of bacterial isolates produced from 100 to 400 mg/dl of histamine in broth.83 The strong histamine formers were M morganii,Proteus spp., and a Raoutella sp., whereas weak formers included H alvei and Proteus spp Skipjack tuna spoiled in seawater at 38◦ C contained C perfringens and V alginolyticus among other histidine decarboxylase producers.117 A strain of M morganii isolated from anchovies was shown to produce 2,377 ± 350 ppm histamine in a culture medium at 37◦ C in 24 hours.91 This strain also produced detectable levels of putrescine and cadaverine From an outbreak of scombroid poisoning associated with tuna sashimi, K pneumoniae was recovered and shown to produce 442 mg/dl of histamine in a tuna fish infusion broth.102 This syndrome has been associated with foods other than scombroid fish, particularly cheeses, including Swiss cheese, which in one case contained 187 mg/dl of histamine; the symptoms associated with the outbreak occurred in 30 minutes to hour after ingestion.103 The number of outbreaks reported to the CDC for the years 1972–1986 were 178 with 1,096 cases but no deaths.26 The largest outbreaks of 51, 29, and 24 occurred in Hawaii, California, and New York, respectively The three most common vehicle foods were mahi mahi (66 outbreaks), tuna (42 outbreaks), and bluefish (19 outbreaks) Although fresh fish normally contains mg/dl of histamine, some may contain up to 20 mg/dl, a level that may lead to symptoms in some individuals The FDA hazardous level for tuna is 50 mg/dl.26 The cooking of toxic fish may not lead to safe products The histamine content of stored skipjack tuna can be estimated if incubation times and temperatures of storage are known Frank et al.37 found that 100 mg/dl formed in 46 hours at 70◦ F, in 23 hours at 90◦ F, and in 17 hours at 100◦ F A nomograph was constructed over the temperature range of 70–100◦ F, underscoring the importance of low temperatures in preventing or delaying histamine formation Vacuum packaging is less effective than low-temperature storage in controlling histamine production.114 The culture medium of choice for detecting histamine-producing bacteria is that of Niven et al.82 Histamine production is favored by low pH, but it occurs more when products are stored above the refrigerator range The lowest temperature for production of significant levels was found to be 30◦ C for H alvei, C freundii, and E coli; and 15◦ C for two strains of M morganii.6 The syndrome is contracted by eating fresh or processed fish of the type noted; symptoms occur within minutes and for up to hours after ingestion of toxic food, with most cases occurring within hour Typical symptoms consist of a flushing of the face and neck accompanied by a feeling of intense heat and general discomfort, and diarrhea Subsequent facial and neck rashes are common The flush is followed by an intense, throbbing, headache tapering to a continuous dull ache Other symptoms include dizziness, itching, faintness, burning of the mouth and throat, and the inability to swallow.50 The minimum level of histamine thought necessary to cause symptoms is 100 mg/dl Large numbers of M morganii in fish of the type incriminated in this syndrome and a level of histamine more than 10 mg/dl is considered significant relative to product quality The first 50 incidents in Great Britain occurred between 1976 and 1979, with all but 19 occurring in 1979 Canned and smoked mackerel was the most common vehicle, with bonita, sprats, and pilchards involved in one outbreak each The most common symptom among the 196 cases was diarrhea.43 734 Modern Food Microbiology Regarding etiology, Hudson and Brown50 believe the evidence does not favor histamine per se as the agent responsible for the syndrome They suggest a synergistic relationship involving histamine and other as yet unidentified agents such as other amines or factors that influence histamine absorption This view is based on the inability of large oral doses of histamine or histamine-spiked fish to produce symptoms in volunteers On the other hand, the suddenness of onset of symptoms is consistent with a histamine reaction, and the association of the syndrome with scombroid fish containing high numbers of histidine–decarboxylase-producing bacteria cannot be ignored Although the precise etiology may yet be in question, bacteria play a significant if not indispensable role Aeromonas This genus consists of several species that are often found in gastrointestinal specimens Among these are A caviae, A eucrenophila, A schubertii, A sobria, A veronii, and A hydrophila An enterotoxin has been identified in A caviae76 and A hydrophila (see below), and the other species noted are associated with diarrhea As A hydrophila has received the most study, the discussion that follows is based on this species The aeromonads are basically aquatic forms that are often associated with diarrhea, but their precise role in the etiology of gastrointestinal syndromes is not clear A hydrophila is an aquatic bacterium found more in salt waters than in fresh waters It is a significant pathogen to fish, turtles, frogs, snails, and alligators and is also a human pathogen, especially in compromised hosts It is a common member of the bacterial biota of pigs Diarrhea, endocarditis, meningitis, soft-tissue infections, and bacteremia are caused by A hydrophila Virulent strains of A hydrophila produce a 52-kDa single polypeptide that possesses enterotoxic, cytotoxic, and hemolytic activities This multifunctional molecule displays immunological crossreactivity with the cholera toxin.92 According to some investigators,116 it resembles aerolysin while others contend that it is aerolysin.78 Aerolysin is a pore- or channel-forming toxin that kills cells by forming discrete channels in their plasma membranes.12 Ion channels are created by the oligomerization of toxin molecules Cytotonic activity has been associated with an A hydrophila toxin, which induced rounding and steroidogenesis in Y-1 adrenal cells Also, positive responses in the rabbit ileal loop, suckling mouse, and CHO assays have been reported for a cytotonic toxin.27 A large number of studies have been conducted on A hydrophila isolates from various sources In one study, 66 of 96 (69%) isolates produced cytotoxins, whereas 32 (80%) of 40 isolates from diarrheal disease victims were toxigenic, with only 41% of nondiarrheal isolates being positive for cytotoxin production Most enterotoxigenic strains are VP (Voges-Proskauer test) and hemolysin positive and arabinose negative13 and produce positive responses in the suckling mouse, Y-1 adrenal cell, and rabbit ileal loop assays In a study of 147 isolates from patients with diarrhea, 91% were enterotoxigenic, whereas only 70% of 94 environmental strains produced enterotoxin as assessed by the suckling mouse assay.14 All but four of the clinical isolates produced hemolysis of rabbit red blood cells Of 116 isolates from the Chesapeake Bay, 71% were toxic by the Y-1 adrenal cell assay, and toxicity correlated with lysine decarboxylase and VP reactions.56 In yet another study, 48 of 51 cultures from humans, animals, water, and sewage, produced positive responses in rabbit ileal loop assays with 103 or more cells, and cell-free extracts from all were loop positive.3 Isolates from meat and meat products have been shown to possess biochemical markers that are generally associated with toxic strains of other species, with the mouse median lethal dose (LD50 ) being log 8–9 colony-forming units (cfu) for most strains tested.85 The latter investigators suggested the possibility that immunosuppressive states are important factors in food-associated infections by this organism, a suggestion that could explain the difficulty of establishing this organism as the sole etiological agent of foodborne gastroenteritis Viruses and Some Other Proven and Suspected Foodborne Biohazards 735 With regard to growth temperature and habitat, of 13 strains displayed growth at 0–5◦ C, of 13 at 10◦ C, and at a minimum of 15◦ C.93 The psychrotrophs had optimum growth between 15◦ C and 20◦ C The maximum growth temperature for some strains was 40–45◦ C with optimum at 35◦ C.46 Regarding distribution, the organism was found in all but 12 of 147 lotic and lentic habitats.46 Four of those habitats that did not yield the organism were either hypersaline lakes or geothermal springs Some waters contained up to 9,000/ml An ecological study of A hydrophila in the Chesapeake Bay revealed numbers ranging from < 0.3/l–5 × 103 /ml in the water column, and about 4.6 × 102 /g of sediment.56 The presence of this organism correlated with total, aerobic, viable, and heterotrophic bacterial counts, and its presence was inversely related to dissolved O2 and salinity, with the upper salt level being about 15% Fewer were found during the winter than during the summer months For reviews, see references 2, 52 Their occurrence in some ready-to-eat foods is presented in Chapter (Table 9–3) Plesiomonas P shigelloides is found in surface waters and soil and has been recovered from fish, shellfish, other aquatic animals, as well as from terrestrial meat animals It differs from A hydrophila in having G + C content of DNA of 51%, versus 58–62% for A hydrophila It has been isolated by many investigators from patients with diarrhea and is associated with other general infections in humans It produces a heat-stable enterotoxin, and serogroup 0:17 strains react with Shigella group D antisera.1 In a study of 16 strains from humans with intestinal illness, P shigelloides did not always bind Congo red, the strains were noninvasive in HEp-2 cells, and they did not produce Shiga-like toxin on Vero cells.1 Although a low-level cytolysin was produced consistently, the mean LD50 for outbred Swiss mice was 3.5 × 108 cfu Heat-stable enterotoxin was not produced by any of the 16 strains, and it was the conclusion of these investigators that this organism possesses a low pathogenic potential.1 P shigelloides was recovered by Zajc-Satler et al.118 from the stools of six diarrheal patients It was believed to be the etiological agent, although salmonellae were recovered from two patients Two outbreaks of acute diarrheal disease occurred in Osaka, Japan, in 1973 and 1974, and the only bacterial pathogen recovered from stools was P shigelloides In the 1973 outbreak, 978 of 2,141 persons became ill, with 88% complaining of diarrhea, 82% of abdominal pain, 22% of fever, and 13% of headaches.107 The symptoms lasted to days Of 124 stools examined, 21 yielded P shigelloides 017:H2 The same serovar was recovered from tap water In the 1974 outbreak, 24 of 35 persons became ill with symptoms similar to those noted P shigelloides serovar 024:H5 was recovered from three of eight stools “virtually in pure culture.”107 The organism was recovered from 39% of 342 water and mud samples, as well as from fish, shellfish, and newts A 15-year-old female contracted gastroenteritis, and hours after she took one tablet of trimethoprim-sulfadiazine, P shigelloides could be recovered from her blood.87 The latter investigators noted that 10 of the previously known 12 cases of P shigelloides bacteremia were in patients who were either immunocompromised or presented with other similar conditions The 15-year-old had a temperature of 39◦ C and passed up to 10 watery stools daily The isolated strain reacted with S dysenteriae serotype antiserum, placing it in O group 22 of P shigelloides.83 Growth of P shigelloides has been observed at 10◦ C,93 and 59% of 59 fish from Zaire waters contained the organism.111 In the latter study, the organism was found more in river fish than lake fish It appeared not to produce an enterotoxin since only of 29 isolates produced positive responses in rabbit ileal loops.95 Foodborne cases have not been documented, but the organism has been incriminated in at least two outbreaks.73 736 Modern Food Microbiology Bacteroides fragilis This obligately anaerobic, Gram-negative bacterium is of potential significance as a foodborne pathogen since it produces an ileal loop-positive enterotoxin and is often associated with human diarrhea, as are A hydrophila and P shigelloides The enterotoxin was first demonstrated in 1984, and enterotoxic strains of B fragilis were first associated with human diarrhea in 1987 B fragilis is estimated to constitute between 1% and 2% of the human intestinal biota As a non-pore former, it is more sensitive to aerated environments than the clostridia and yet it has been recovered from municipal sewage This species differs from most other Bacteroides in being catalase positive, and like most others it can grow in the presence of 20% bile The B fragilis enterotoxin is produced as a single chain with a molecular weight of about 20,000 Da It differs from the classic bacterial enterotoxins in belonging to a class of zinc-binding metalloprotease, designated metzincins The enterotoxin has a wide range of protein substrates, and it undergoes autodigestion The intestinal damage that it causes is believed to be due, at least in part, to its proteolytic action It elicits a positive response in ileal loops of lambs and other animals Since the etiological agent is identified in only around 50% of foodborne outbreaks in the United States, it is clear that previously unrecognized agents need to be included B fragilis along with Klebsiella pneumoniae60 and Enterobacter cloacae61 may warrant more attention The latter two organisms produce heat-stable enterotoxins that are similar to the heat-stable enterotoxin (ST) of E coli, and their potential significance in foods has been noted.109 Erysipelothrix rhusiopathiae This bacterium (E·ry·si·pe’·lo·thrix rhu·si·o·pa’·thi·ae) is phylogenetically closely related to Listeria (see Chapter 25), and like L monocytogenes, it causes disease in animals and humans It is the cause of erysipelas in swine and erysipeloid in humans Because of these similarities, it seems to be a “logical” candidate for a foodborne pathogen although such cases not seem to have been reported In general, erysipeloid is a localized disease of the hands and arms of handlers of fresh meat and fish, but systemic involvements are not unknown Erysipeloid in pigs is characterized by diamond-skin lesions The organism is a facultative anaerobe, catalase negative (in contrast to the listeriae), oxidase negative, and generally produces H2 S At least 23 serovars are known The only other species is E tonsillarum, which was separated from E rhusiopathiae based on its primary habitat of porcine tongues, and because of serovar differences.100 One of the first studies of the incidence of this organism in foods is that of Ternstrăom and Molin,104 who in 1982 undertook a study of foodborne pathogens in meats in Sweden They examined 135 samples consisting of equal numbers of chicken, beef, and pork, and found E rhusiopathiae in 36% and 13%, respectively, of pork and chicken, but none in beef In one plant, 54% of pork loins were positive, and many of the isolates possessed mouse virulence Of 112 retail pork samples examined in Japan, 34% contained this bacterium, and the 38 isolates represented 14 serovars.98 In a study of meat samples from 93 wild boar and 36 deer in Japan, 44% of the wild boar and 50% of the deer samples contained E rhusiopathiae, representing 13 serovars.54 In a study of 750 chickens in Japan, Erysipelothrix spp were recovered from 15.7% of skin samples, and from 59.2% of 179 feather samples.76 E rhusiopathiae represented 273 of 297 isolates and the remainder were E tonsillarum In another study of 153 chicken samples in Japan, 30% contained Erysipelothrix spp with 65 of 67 being E rhusiopathiae.77 Viruses and Some Other Proven and Suspected Foodborne Biohazards 737 Klebsiella pneumoniae About hours after consuming a fast-food chain hamburger, an individual complained of not feeling well After hospital admission, this organism along with generic E coli was isolated from leftover hamburger and from the patient’s blood, and the two matched by cultural methods.94 The strain of K pneumoniae was LT+ and ST− The coliform count in leftover hamburger was 3.0 × 106 /g, and 1.9 × 105 /g of bun Streptococcus iniae There have been at least six human infections by this organism traced to a fish product S iniae was first recognized in 1972 as the cause of a disease in Amazon dolphins.20 It was next recorded in Israel in 1986 as the cause of disease in tilapia and trout, and later seen in Taiwan and the United States.89 The first human case was recorded in 1991 in Texas, and the second in 1994 in Ottawa.20 Four human cases occurred in Ontario, Canada, in 1995–1996 and the organism was isolated from both fish and patients The fish was tilapia that was imported from fish farms in the United States S iniae appears to be a fish pathogen that causes disease in humans In the Ontario cases, it appeared that the organism entered the body through hand lesions It is beta-hemolytic on sheep blood In humans, the organism produces fulminant soft tissue infections.39 PRION DISEASES Prions are unique proteins in that they can convert other proteins into damaging ones by causing them to alter their shape The normal cell prion protein (PrP) exists in the brain cell membrane where it carries out some vital functions and is then degraded by proteases However, the pathogenic form is distorted and is resistant to proteases, and thus it accumulates in brain tissue and gives rise to disease (see below) It has been postulated that the distorted prion molecule, acting as a template, converts normal protein to a distorted form.9 The normal protein (α-helical form) takes on a protease-resistant β-flat form (PrPSc , PrPres) when it becomes pathogenic The pathogenic forms tend to aggregate into amyloid fibrils where they cause nerve cell degeneration, which leads to clinical signs of disease Although the evidence of a prion etiology of these diseases seems strong, the possibility that a virus is the agent has been raised.28 These particles were named around 1982 by Stanley Prusiner, who was awarded the 1997 Nobel Prize in physiology for his pioneering work.112 Prions cause the disease scrapie in sheep, goats, and hamsters; and kuru in humans Another prion disease of humans is Creutzfeldt-Jakob disease (CJD) Bovine spongiform encephalopathy (BSE) is a prion disease of cattle and sheep referred to as “mad cow disease.” All of these belong to a family of diseases called transmissible spongiform encephalopathies (TSEs) Bovine spongiform encephalopathy (BSE) BSE (“mad cow disease”) was first recognized in Great Britain in 1984 and specifically diagnosed in cattle in 1986 Four years later, over 14,000 confirmed cases out of a population of 10 million cattle had been recognized in Great Britain The epidemic seemed to peak around 1,000 new cases per week in 1993 By February 1998, a total of 172,324 cases were seen in cattle in the United Kingdom.9 A total ... isolates produced positive responses in rabbit ileal loops.95 Foodborne cases have not been documented, but the organism has been incriminated in at least two outbreaks.73 736 Modern Food Microbiology. .. either immunocompromised or presented with other similar conditions The 15-year-old had a temperature of 39◦ C and passed up to 10 watery stools daily The isolated strain reacted with S dysenteriae... identi? ?ed in A caviae76 and A hydrophila (see below), and the other species noted are associated with diarrhea As A hydrophila has received the most study, the discussion that follows is based on