728 Modern Food Microbiology for viral hepatitis of food origin Just as nonintestinal bacteria of human origin are sometimes found in foods, the same may be true for viruses, but because of their tissue affinities, foods would serve as vehicles only for the intestinal or enteroviruses These agents may be accumulated by some shellfish up to 900-fold.41 Viral gastroenteritis is believed to be second only to the common cold in frequency Incidence in Foods and the Environment A common food source of gastroenteritis-causing viruses is shellfish Although crustaceans not concentrate viruses, molluskan shellfish because they are filter feeders When poliovirus was added to waters, blue crabs were contaminated, but they did not concentrate the virus.47 Shucked oysters artificially contaminated with 104 pfu of a poliovirus retained viruses during refrigeration for 30–90 days with a survival rate of 10–13%.33 It has been reported that the uptake of enteroviruses by oysters and clams is not likely when viruses in the water column are less than 0.01 pfu/ml.66 The recovery method employed by the latter authors was capable of detecting 1.5–2.0 pfu per shellfish Although the coliform index is of proven value as an indicator of intestinal bacterial pathogens in waters, it appears to be inadequate for enteroviruses, which are more resistant to adverse environmental conditions than bacterial pathogens.90 In a study of more than 150 samples of recreational waters from the upper Texas gulf, enteroviruses were detected 43% of the time when by coliform index the samples were judged acceptable, and 44% of the time when judged acceptable by fecal coliform standards.42 In the same study, enteroviruses were found 35% of the time in waters that met acceptable standards for shellfish harvesting, and the investigators concluded that the coliform standard for waters does not reflect the presence of viruses From a study of hard-shell clams off the coast of North Carolina, enteric viruses were found in those from open and closed beds.113 (Closed waters are those not open to commercial shellfishing because of coliform counts.) From open beds, of 13 100-g samples were positive for viruses, whereas all 13 were negative for salmonellae, shigellae, or yersiniae From closed waters, of 15 were positive for salmonellae, and all were negative for shigellae and yersiniae.113 The latter investigators found no correlation between enteric viruses and total coliforms or fecal coliforms in shellfish waters, or total coliforms, fecal coliforms, “fecal streptococci,” or aerobic plate counts (APC) in clams Although enteric viruses may be found in shellfish from open waters, less than 1% of shellfish samples examined by the Food and Drug Administration (FDA) contained viruses.67 (See Chapter 20 for further discussion of safety indicators and intestinal viruses.) With respect to the capacity of certain viruses to persist in foods, it has been shown that enteroviruses persisted in ground beef up to days at 23◦ C or 24◦ C and were not affected by the growth of spoilage bacteria.48 In a study of 14 vegetable samples for the existence of naturally occurring viruses, none were found, but coxsackievirus B5 inoculated onto vegetables did survive at 4◦ C for days.62 In an earlier study, these investigators showed that coxsackievirus B5 had no loss of activity when added to lettuce and stored at 4◦ C under moist conditions for 16 days Several enteric viruses failed to survive on the surfaces of fruits, and no naturally occurring viruses were found in nine fruits that were examined.63 Echovirus and poliovirus were found in each of 17 samples of raw oysters examined by Fugate et al.,38 and poliovirus was found in of 24 samples of oysters Of seven food-processing plants surveyed for human viruses, none was found in a vegetable-processing plant or in three that processed animal products.64 The latter investigators examined 60 samples of market foods but were unable to detect viruses in any They concluded that viruses in the U.S food supply are very low Destruction in Foods The survival of the hog cholera (HCV) and African swine fever viruses (ASFV) in processed meats was studied by McKercher et al.72 From pigs infected with these viruses, partly cooked canned hams Viruses and Some Other Proven and Suspected Foodborne Biohazards 729 and dried pepperoni and salami sausages were prepared; whereas virus was not recovered from the partly cooked canned hams, they were recovered from hams after brining but not after heating The ASFV retained viability in the two sausage products following the addition of curing ingredients and starters but were negative after 30 days HCV also survived the addition of curing ingredients and starter and retained viability even after 22 days The effect of heating on destruction of the foot-and-mouth virus was evaluated by Blackwell et al.8 When ground beef was contaminated with virus-infected lymph node tissue and processed to an internal temperature of 93.3◦ C, the virus was destroyed However, in cattle lymph node tissue, the virus survived for 15 but not 30 minutes at 90◦ C In another study, the thermal destruction of foot-andmouth disease virus (FMDV) along with two others was achieved within minutes at 67◦ C, or at minutes at 60◦ C or 62◦ C in a pig slurry.108 The boiling of crabs was found sufficient to inactivate 99.9% of poliovirus 1, and a rotavirus and an echovirus were destroyed within minutes.47 A poliovirus was found to survive stewing, frying, baking, and steaming of oysters.33 In broiled hamburgers, enteric viruses could be recovered from of 24 patties cooked rare (to 60◦ C internally) if the patties were cooled immediately to 23◦ C.99 No viruses were detected if the patties were allowed to cool for minutes at room temperature before testing Hepatitis A Virus Prior to the 1990s, there were more documented outbreaks of hepatitis A traced to foods than any other viral infection The virus belongs to the family Picornaviridae, as the polio, echo, and coxsackie viruses, and all have single-stranded RNA (ss RNA) genomes The incubation period for infectious hepatitis ranges from 15 to 45 days, and lifetime immunity usually occurs after an attack The fecal–oral route is the mode of transmission, and raw or partially cooked shellfish from polluted waters is the most common vehicle food In the United States in 1973, 1974, and 1975, there were 5, 6, and outbreaks, respectively, with 425, 282, and 173 cases The 1975 outbreaks were traced to salad, sandwiches, and glazed doughnuts served in restaurants The recorded outbreaks and cases in the United States for 1983–1987 are presented in Table 31–1 According to the Centers for Disease Control and Prevention (CDC), hepatitis A increased in the United States between 1983 and 1989 by 58%—from 9.2 to 14.5 per 100,000 persons.25 Of the Table 31–1 Outbreaks, Cases, and Deaths Associated with Viral Foodborne Gastroenteritis in the United States, 1983–1987 Outbreaks/Cases/Deaths Year Hepatitis A Norwalk Agent Other Viruses 1983 1984 1985 1986 1987 Totals 10/530/1 2/29/0 5/118/0 3/203/0 9/187/0 29/1067/1 1/20/0 1/137/0 4/179/0 3/463/0 1/365/0 10/1164/0 – 1/444/0 1/114/0 – – 2/558/0 Source: From N.H Bean, P.M Griffin, J.S Goulding, and C.B Ivey, 1990 J Food Protect 53:711–728 730 Modern Food Microbiology cases in 1988, 7.3% were either foodborne or waterborne.25 Among 88 elementary school students and their teachers in 1990 in the state of Georgia, 15 came down with hepatitis A Among 641 residents and staff at an institution for the disabled in Montana, 13 contracted hepatitis A Strawberry shortcake was the vehicle in both outbreaks The frozen strawberries came from the same processing plant in California.81 The largest foodborne outbreak of hepatitis A ever recorded in the United States occurred in November 2003, and there were ca 600 victims with deaths The vehicle food was imported green onions (scallions) served by a fast-food restaurant chain Between 1992 and 2001, an estimated 230,000 cases of hepatitis A were reported to the CDCP In 2001, an outbreak of >46 cases occurred in Massachusetts and it was associated with the consumption of sandwiches, which apparently were contaminated by a food handler.16 Noroviruses The former Norwalk, Norwalk-like,and small-round-structured viruses (SRSV) have been placed in this group as the genus Norovirus of the human caliciviruses (HuCV) The noroviruses are in two genogroups, I and II The former Norwalk group is the prototype for genogroup I, and the former Snow Mounain viruses are in genogroup II All of these viruses are unenveloped and ssRNA with a diameter of 27–40 nm and their genome consists of 7,300 to 8,300 base pairs The caliciviruses also include the genus Sapovirus For more information, see references 40, 86, 96 The Norwalk virus was first recognized in a school outbreak in Norwalk, Ohio in 1968, and water was suspected, but not proven, as the source It is the most prevalent of the noroviruses in foods The virus is more resistant to destruction by chlorine than other enteric viruses In volunteers, 3.75 ppm chlorine in drinking water failed to inactivate the virus, whereas poliovirus type and human and simian rotaviruses were inactivated.58 Some Norwalk viruses remained infective at residual chlorine levels of 5–6 ppm Hepatitis A viruses are not as resistant as Norwalk, but both are clearly more resistant to chlorine than the rotaviruses The exposure of noroviruses to 0.37 mg/l of ozone at pH and 5◦ C for up to minutes in water effected a >3 log10 reduction after a 10 second exposure.97 Of 430 foodborne outbreaks in the United States in 1979, 4% displayed the pattern of Norwalk gastroenteritis.57 This virus was thought to be the cause of more foodborne gastroenteritis than any single bacterium in the state of Minnesota in 1985.65 Noroviruses are now the leading cause of gastroenteritis in the United States with an estimated 23 million cases per year.17 From several cruise ship outbreaks in 2002, there were at least 1,786 victims Up to 21 ship outbreaks occurred during the same general time period, and the virus appeared to come from unspecified environmental sources Of 1,412 outbreaks of foodborne intestinal diseases in England and Wales for the years 1992–1999, 82 (5.8%) were caused by noroviruses (all previously designated SRSVs) From the same survey, 12 of 60 (20%) were traced to fruits and vegetables.96 Since they have yet to be cultured in the laboratory, the method of choice is RT–PCR (see Chapter 11) During the fall of 2001, a rare waterborne outbreak of norovirus gastroenteritis occurred in the state of Wyoming, and there were around 84 victims Ground water was contaminated with sewage, which was the source of the human HuCV The etiologic agents were identified by use of RT–PCR, which demonstrated mainly genogroup I, subtype 3; but one stool sample yielded a genogroup II, subtype strain.86 Among the earliest reported outbreaks that involved noroviruses is one that occurred in 1976 in England Between December 21, 1976, and January 10, 1977, 33 outbreaks and 797 cases occurred, Viruses and Some Other Proven and Suspected Foodborne Biohazards 731 and cockles were incriminated.4 The incubation period was 24–30 hours, and from 12 of 14 stool samples, small, round virus particles measuring 25–26 nm in diameter were demonstrated, but they were not found in cockles Although the investigators believed that the agents were neither Norwalk nor Hawaii, these outbreaks are regarded by some as Norwalk virus outbreaks The 1978 outbreak in Australia that involved at least 2,000 persons was well documented, and the vehicle food was oysters.74 The virus was found in 39% of fecal specimens examined by electron microscopy, and antibody responses were demonstrated in 75% of paired sera tested The incubation period ranged from 18 to 48 hours, with most cases occurring in 34–38 hours Nausea was the first symptom, usually accompanied by vomiting, nonbloody diarrhea, and abdominal cramps, with symptoms lasting 2–3 days Another outbreak in Australia was traced to bottled oysters and symptoms occurred in 24–48 hours.36 The oysters had an APC of 2.2 × 104 /g and a fecal coliform count of 500/100 g The first documented food source outbreaks in the United States are those that occurred in New Jersey in 1979, where lettuce was the vehicle food, and the Florida outbreak in 1980 that was traced to raw oysters In the latter, the agent was identified by a radioimmunoassay method Rotaviruses The first demonstration of these viruses occurred in 1973 in Australia, and they were first propagated in the laboratory in 1981 Six groups have been identified, and three are known to be infectious for humans Group A is the most commonly encountered among infants and young children throughout the world Group B causes diarrhea in adults, and they have been seen only in China Rotaviruses belong to the family Reoviridae; they are about 70 nm in diameter, are nonenveloped, and contain double-stranded RNA (dsRNA) The fecal–oral route is the primary mode of transmission Rotaviruses cause an estimated one-third of all hospitalizations for diarrhea in children below age 5, and the peak season for infection occurs during the winter months Most susceptible are children between the ages of months and years, and it has been reported that virtually every child in the United States is infected by age 4.24 Although most persons are immune by age 4, high inoculum or lowered states of immunity can lead to milder illness among older children and adults.24 These viruses are known to be transmitted among children in day care centers and by water A community waterborne outbreak occurred in Eagle-Vail, Colorado, in 1981, and 44% of 128 persons, most of them adults, became ill.49 They are believed to be only infrequent causes of foodborne gastroenteritis.30 The incubation period for rotavirus gastroenteritis is days Vomiting occurs for days accompanied by watery diarrhea for 3–8 days, and often abdominal pain and fever also occur.24 They are known to be associated with travelers’ diarrhea It appears that these viruses induce diarrhea by activating the enteric nervous system (ENS) based on the inhibition of ENS functions in mice and in vitro by four drugs that inhibit ENS functions.70 For the 23-month period between January 1989 and November 1990, 48,035 stool specimens were examined in the United States, with 9,639 (20%) being positive for rotavirus.22 The highest percentage of positive stools occurred in February (36%) and the lowest in October (6%) Between 1979 and 1985, an annual average of 500 children died from diarrheal illness in the United States, and 20% were caused by rotavirus infections.22 The host-cell-receptor protein for rotavirus also serves as the β-adrenergic receptor Once inside cells, they are transported to lysosomes where uncoating occurs Rotaviral infections can be diagnosed by immunoelectron microscopy, RT–PCR, enzyme-linked immunosorbent assay (ELISA), and latex agglutination methods 732 Modern Food Microbiology BACTERIA Enterobacter sakazakii This bacterium, once classified as a yellow-pigmented Enterobacter cloaceae, has been identified as the cause of neonatal necrotizing enterocolitis (NEC); neonatal meningitis; and sepsis, dating back to 1961 The usual vehicle food is powdered milk formulas Although it is considered to be an opportunistic pathogen, some strains produce an enterotoxin, and they are lethal to suckling mice Citrobacter freundii has been identified as the cause of neonatal infections and the vehicle foods were infant formulas (see reference 105) In one study, of 18 E sakazakii strains produced enterotoxin, and at 108 cfu/mouse by the intraperitoneal route, all 18 isolates were lethal to suckling mice (16–18 days old) and were lethal by the peroral route.84 In addition to suckling mice, potential virulence is manifested in monolayers of CHO, Vero, and Y-1 adrenal cells Infant mortality rates range from 40 to 60% In an outbreak of 12 cases in 1998 in an intensive care unit in Belgium, infants died and E sakazakii was recovered from unused prepared formula and from unopened cans of a single batch.110 In a study of enteric bacteria in 141 powdered milk formulas from 35 countries, 25% contained Pantoea agglomerans, 21% E cloaceae, and 14% E sakazakii.75 From a study of 120 dried infant formulas in Canada, (6.7%) contained E sakazakii.80 The minimum growth temperature of tested isolates in the latter study was 5.5 to 8◦ C, and the maximum growth temperature ranged from 41 to 45◦ C with a mean of 42.5◦ C for 11 isolates No isolates grew at 4◦ C.79 In regards to the thermal resistance of E sakazakii, it appears to be higher than that of most Gramnegative bacteria In a study of 10 strains (5 clinical and food isolates) in reconstituted dried-infant formula, a mean D60◦ C of 2.5 minutes and z = 5.82◦ C were found.80 The mean D60◦ C for the clinical isolates was 2.15, and 3.06 for the food isolates In another study, the D58◦ C of 12 strains in rehydrated infant formula ranged from 30.5 to 591.9 seconds (0.508 to 9.865 minutes).35 By adding the most heat-resistant strain of E sakazakii (z = 5.6◦ C) found in the last study to dry infant formula rehydrated at 70◦ C, > 4-log reduction was achieved.35 According to these investigators, if it is assumed that the typical level of this organism in infant formula is cfu/100 g of dry formula, a 4-D treatment should assure its absence after cooling for infant feeding.35 The heat-resistant strain employed was the most resistant of 12 tested Stationary phase cells of E sakazakii have been reported to be more resistant to osmotic and dry stress than E coli and some other bacteria; and this increased resistance appeared to be associated with the accumulation of trehalose by stationary phase cells.10 The latter investigators found D58◦ C values of 0.27–0.50 for E sakazakii strains compared to 0.40–0.50 for three salmonellae Histamine-Associated (Scombroid) Poisoning Illness contracted from eating scombroid fish or fish products containing high levels of histamine is often referred to as scombroid poisoning Among the scombroid fishes are tuna, mackerel, bonito, and others In one report, histamine poisoning was associated with sailfish, a nonscombroid.51 The histamine is produced by bacterial decarboxylation of the generally large quantities of histidine in the muscles of this group Sufficient levels of histamine may be produced without the product being organoleptically unacceptable, with the result that scombroid poisoning may be contracted from both fresh and organoleptically spoiled fish The history of this syndrome has been reviewed by Hudson and Brown,50 who questioned the etiological role of histamine This is discussed further below ... and Suspected Foodborne Biohazards 729 and dried pepperoni and salami sausages were prepared; whereas virus was not recovered from the partly cooked canned hams, they were recovered from hams... A ever recorded in the United States occurred in November 2003, and there were ca 600 victims with deaths The vehicle food was imported green onions (scallions) served by a fast -food restaurant... 1975 outbreaks were traced to salad, sandwiches, and glazed doughnuts served in restaurants The recorded outbreaks and cases in the United States for 1983–1987 are presented in Table 31–1 According