668 Modern Food Microbiology of nonenterocolitica species, including Y intermedia and Y kristensenii, were positive for ST in two other studies.129,136 Y bercovieri produces a heatstable enterotoxin (YbST), and detectable levels are produced at 4◦ C after 144–168 hours.122 Although pathogenic strains of Y enterocolitica produce ST, it appears that this agent is not critical to virulence Some evidence for the lack of importance of ST was provided by Schiemann,112 who demonstrated positive HeLa-cell and Sereny-test responses, with a 0:3 strain that did not produce enterotoxin On the other hand, each of 49 isolates belonging to serovar 0:3 and the other four virulent serovars produced ST.88 In addition to the diminished role of ST in Y enterocolitica virulence, some other properties now seem less important.22,81 The Yop virulon is the most significant virulence factor for yersiniae, and it along with some of the more recent findings on the pathogenesis of these organisms is discussed in Chapter 22 Incidence of Y enterocolitica in Foods This organism has been isolated from cakes, vacuum-packaged meats, seafood, vegetables, milk, and other food products It has been isolated also from beef, lamb, and pork.77 Of all sources, swine appears to be the major source of strains pathogenic for humans More specifically, pig tonsils have been found to be the primary sources of contamination of liver, heart, and kidneys.43 The incidence and growth of Y enterocolitica in milk are presented in Chapter 7, and for meats in Chapter Gastroenteritis Syndrome and Incidence In addition to gastroenteritis, this organism has been associated with human pseudoappendicitis, mesenteric lymphadenitis, terminal ileitis, reactive arthritis, peritonitis, colon and neck abscesses, cholecystis, and erythema nodosum It has been recovered from urine, blood, cerebrospinal fluid, and the eyes of infected individuals It is, of course, recovered from the stools of gastroenteritis victims Only the gastroenteritis syndrome is addressed below There is a seasonal incidence associated with this syndrome, with the fewest outbreaks occurring during the spring and the greatest number in October and November The incidence is highest in the very young and the old In an outbreak studied by Gutman et al.,49 the symptoms (and percentage complaining of them) were fever (87), diarrhea (69), severe abdominal pain (62), vomiting (56), pharyngitis (31), and headache (18) The outbreak led to two appendectomies and two deaths Milk (raw, improperly pasteurized, or recontaminated) is a common vehicle food The first documented outbreak in the United States occurred in 1976 in New York State, with serovar 0:8 as the responsible strain, and chocolate milk prepared by adding chocolate syrup to previously pasteurized milk was the vehicle food.12 An outbreak of serotype 0:3 among 15 children occurred in Georgia in 1988–1989; the vehicle food was raw chitterlings.30 Symptoms of the gastroenteritis syndrome develop several days following ingestion of contaminated foods and are characterized by abdominal pain and diarrhea Children appear to be more susceptible than adults, and the organisms may be present in stools for up to 40 days following illness.4 A variety of systemic involvements may occur as a consequence of the gastroenteritis syndrome CAMPYLOBACTERIOSIS (Campylobacter ampylobacter jejuni) jejuni The genus Campylobacter consists of at least species, and the one of primary importance in foods is C jejuni subsp jejuni Unlike C jejuni subsp doylei, it is resistant to cephalothin, can grow at 42◦ C, and can reduce nitrates Throughout this text, C jejuni subsp jejuni is referred to as C jejuni Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 669 The latter differs from Campylobacter coli in being able to hydrolyze hippurate The campylobacters are more closely related to the genus Arcobacter than any other group C jejuni has the distinction of being the first foodborne pathogen whose genome was sequenced (it contains 1.64 million bases) Prior to the 1970s, the campylobacters were known primarily to veterinary microbiologists as organisms that caused spontaneous abortions in cattle and sheep and as the cause of other animal pathologies They were once classified as Vibrio spp C jejuni is a slender, spirally curved rod that possesses a single polar flagellum at one or both ends of the cell It is oxidase and catalase positive and will not grow in the presence of 3.5% NaCl or at 25◦ C It is microaerophilic, requiring small amounts of oxygen (3–6%) for growth Using an autobioluminescent strain of C jejuni, its minimum, optimum, and maximum growth temperatures on solid media in a gradient-plate format were 30, 40, and 45◦ C.68 At the optimum growth temperature of 40◦ C, this strain grew well at pH 5.5–8.0, and in the presence of up to 1.75% NaCl Growth is actually inhibited in 21% oxygen Carbon dioxide (about 10%) is required for good growth When C jejuni was inoculated into vacuum-packaged processed turkey meat, cell numbers decreased, but some remained viable for 28 days at 4◦ C.105 Its metabolism is respiratory In addition to C jejuni, C coli, C intestinalis, and several other Campylobacter species are known to cause diarrhea in humans, but C jejuni is by far the most important Because of their small cell size, they can be separated from most other Gram-negative bacteria by use of a 0.65-µm filter C jejuni is heat sensitive, with D55◦ C for a composite of equal numbers of five strains being 1.09 minutes in peptone and 2.25 minutes in ground, autoclaved chicken.14 With internal heating of ground beef to 70◦ C, 107 cells/g could not be detected after about 10 minutes.120 It appears to be sensitive to freezing, with about 105 cells per chicken carcass being greatly reduced or eliminated at -18◦ C, and for artificially contaminated hamburger meat, the numbers were reduced by log cycle over a 7-day period.47 Distribution Unlike Y enterocolitica and V parahaemolyticus, C jejuni is not an environmental organism but rather is one that is associated with warm-blooded animals A large percentage of all major meat animals have been shown to contain these organisms in their feces, with poultry being prominent Its prevalence in fecal samples often ranges from around 30% to 100% Reports on isolations by various investigators have been summarized by Blaser,15 and the specimens and percentages positive for C jejuni are as follows: chicken intestinal contents (39–83), swine feces (66–87), sheep feces (up to 73), swine intestinal contents (61), sheep carcasses (24), swine carcasses (22), eviscerated chicken (72–80), and eviscerated turkey (94) In a 5-year longitudinal study on a small rearing farm in southern England in 1989–1994, 12,233 broilers were examined and 27% were positive for C jejuni.102 Of 251 shed flocks, 35.5% carried C jejuni, but only 9.2% had the organism in successive flocks Overall, there was a low level of transmission between flocks A common source was suggested via vertical transmission rather than hatchery or transportation sources because of the lack of high diversity of types.102 The ecology and prevalence of campylobacters in other fresh foods and the environment have been reviewed.91 Fecal specimens from humans with diarrhea yield C jejuni, and it may be the single most common cause of acute bacterial diarrhea in humans From 8,097 specimens submitted to eight hospital laboratories over a 15-month period in different parts of the United States, this organism was recovered from 4.6%, salmonellae from 2.3%, and shigellae from 1%.16 The peak isolations for C jejuni were in the age group 10–29 years Peak isolations occur during the summer months, and it has been noted that 3–14% of diarrheal patients in developed countries yield stool specimens that contain C jejuni.15 670 Modern Food Microbiology Peak isolations from individually caged hens occurred in October and late April–early May.39 In the latter study, 8.1% of the hens were chronic excreters of the organism, whereas 33% were negative even though they were likely exposed The most probable source of C jejuni to a duck processing farm was found to be rat and mice droppings, with 86.7% of the former being positive for this species.67 The consensus seems to be that this organism is not transmitted through the hatchery, but instead to broiler chicks by vermin as noted The cecum is the principal site of colonization, and the organisms generally are not pathogenic to adult birds The numbers of C jejuni on some poultry products range from log 2.00 to 4.26/g Once this organism is established in a chicken house, most of the flock becomes infected over time One study revealed that the organism appeared in all chicken inhabitants within a week once it was found among any of the inhabitants.117 In addition to poultry, the other primary source of this organism is raw milk Because the organism exists in cow feces, it is not surprising that it may be found in raw milk, and the degree of contamination would be expected to vary depending on milking procedures In a survey of 108 samples from bulk tanks of raw milk in Wisconsin, only was positive for C jejuni, whereas the feces of 64% of the cows in a grade A herd were positive.40 In the Netherlands, 22% of 904 cow fecal and 4.5% of 904 raw milk samples contained C jejuni.11 The significance of cross-contamination as a source of this organism to humans is illustrated by the following outbreak: There were 14 cases of Campylobacter enteritis in Oklahoma traced to lettuce and lasagna.26 The foods were prepared in a small area where raw chicken was cut, and in all probability, the vehicle foods became contaminated See Chapters and for numbers of campylobacters in various foods Virulence Properties At least some strains of C jejuni produce a heat-labile enterotoxin (CJT) that shares some common properties with the enterotoxins of V cholerae (CT) and E coli (LT) CJT increases cAMP levels, induces changes in CHO cells, and induces fluid accumulation in rat ileal loops.108 Maximal production of CJT in a special medium was achieved at 42◦ C in 24 hours, and the amount produced was enhanced by polymyxin.72 The quantities produced by strains varied widely from none to about 50 ng/ml CJT protein The amount of toxin was doubled as measured by Y-1 adrenal cell assay when cells were first exposed to lincomycin and then polymyxin.83 CJT is neutralized by CT and E coli LT antisera, indicating immunological homology with these two enterotoxins.72 The C jejuni LT appears to share the same cell receptors as CT and E coli LT, and it contains a B subunit immunologically related to the B subunits of CT and LT of E coli.73 Also, a cytotoxin is produced that is active against Vero and HeLa cells The enterotoxin and the cytotoxin induce fluid accumulation in rat jejunal loops but not in mice, pigs, or calves Partially purified enterotoxin contained three fractions with molecular weights of 68, 54, and 43 kDa.61 Of 202 strains of C jejuni and C coli recovered from humans with enteritis and from healthy laying hens, 34% and 22% of the C jejuni and C coli strains, respectively, produced enterotoxin as determined by CHO assay.78 From a comparison of poultry and human isolates of Campylobacter jejuni, invasiveness or Vero cell toxicity was higher for human than for poultry isolates.89 Invasiveness was associated with biotypes and while cell toxicity (with CHO and INT-407 cells) was associated with biotypes and C jejuni enteritis appears to be caused in part by the invasive abilities of the organism Evidence for this comes from the nature of the clinical symptoms, the rapid development of high agglutinin titers after infection, recovery of the organism from peripheral blood during the acute phase of the disease, Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 671 and the finding that C jejuni can penetrate HeLa cells.80 However, C jejuni is not invasive by either the Sereny or the Anton assay Caco-2 cells are invaded by an energy-dependent invasion mechanism, not by endocytosis.109 Several sequelae are associated with campylobacteriosis, including Guillain–Barr´e syndrome (GBS: for a review, see reference 116) It is estimated that about one-third of patients with GBS develop symptoms 1–3 weeks after C jejuni enteritis In the Penner serotyping scheme, over 48 serotypes of C jejuni are recognized, and serotype 19 is one that appears to be associated with GBS This strain has an oligosaccharide structure that is identical to the terminal tetrasaccharide of host ganglioside GM1 Because the gangliosides are surface components of nerve tissue, antibodies to the oligosaccharide structure of C jejuni would exhibit antineural effects.142 Plasmids have been demonstrated in C jejuni cells Of 17 strains studied, 11 were found to carry plasmids ranging from 1.6 to 70 MDa, but their role and function in disease are unclear A serotyping scheme has been developed for C jejuni From chickens and humans, 82% and 98%, respectively, of isolates belonged to biovar 1.113 Overall, the specific modes of Campylobacter pathogenesis are still unclear In one review, it is noted that motility and invasion play a role in pathogenesis, and that the roles of the toxins are far from clear.71 Enteritis Syndrome and Prevalence From the first U.S outbreak of C jejuni traced to a water supply,29 in which about 2,000 individuals contracted infections, the symptoms (and percentages of individuals affected) were as follows: abdominal pain or cramps (88), diarrhea (83), malaise (76), headache (54), and fever (52) Symptoms lasted from to days In the more severe cases, bloody stools may occur, and the diarrhea may resemble ulcerative colitis, whereas the abdominal pain may mimic acute appendicitis.15 The incubation period for enteritis is highly variable It is usually 48–82 hours but may be as long as 7–10 days or more Diarrhea may last 2–7 days, and the organisms may be shed for more than months after symptoms subside Clinical laboratory isolates of Campylobacter in the United States for 1996–1998 exceeded salmonellae isolates (Figure 28–1) The isolates were from clinical laboratories in selected cities of five states This is the FoodNet Surveillance Network24 described in Chapter 22 It should be noted that the numbers not represent isolations from actual foodborne illness It is assumed that the organisms noted were contracted from foods even though this connection is not demonstrated In the case of Campylobacter, it is assumed that about 90% are of food origin While this surveillance method might be a valid indicator of actual foodborne cases, it is unprecedented It is unusual that such a fragile and environmentally sensitive organism should be the leading cause of foodborne illness It is interesting to note that the largest recorded outbreak of Campylobacter enteritis, as noted above, was traced to the water supply of a Vermont town where about 2,000 persons were infected.29 PREVENTION V parahaemolyticus, Y enterocolitica, and C jejuni are all heat-sensitive bacteria that are destroyed by milk pasteurization temperatures The avoidance of raw seafood products and care in preventing cross-contamination with contaminated raw materials will eliminate or drastically reduce the incidence of foodborne gastroenteritis caused by V parahaemolyticus and Y enterocolitica To prevent wound 672 Modern Food Microbiology Figure 28–1 Rate (per 100,000 population) of laboratory-confirmed infections with selected pathogens detected by the Foodborne Diseases Active Surveillance Network (FoodNet), United States, 1966–1998 The results for 1998 are preliminary 1999 Morbidity and Mortality Weekly Report 48:190 U S Center for Disease Control and Prevention infections by vibrios, individuals with body nicks or abrasions should avoid entering estaurine or seawaters Yersinosis can be avoided or certainly minimized by not drinking water that has not been properly treated, and by avoiding raw or underprocessed milk Campylobacteriosis can be avoided by not eating undercooked or unpasteurized foods of animal origin, especially milk and raw poultry REFERENCES Abbott, S.L., C Powers, C.A Kaysner, Y Takeda, M Ishibashi, S.W Joseph, and J.M Janda 1989 Emergence of a restricted bioserovar of Vibrio parahaemolyticus as the predominant cause of Vibrio-associated gastroenteritis on the West Coast of the United States and Mexico J Clin Microbiol 27:2891–2893 Amin, M.K., and F.A Draughon 1987 Growth characteristics of Yersinia enterocolitica in pasteurized skim milk J Food Protect 50:849–852 Amirmozafari, N., and D.C Robertson 1993 Nutritional requirements for synthesis of heat-stable enterotoxin by Yersinia enterocolitica Appl Environ Microbiol 59:3314–3320 Asakawa, Y., S Akahane, N Kagata, and M Noguchi 1973 Two community outbreaks of human infection with Yersinia enterocolitica J Hyg 71:715–723 Baross, J., and J Liston 1970 Occurrence of Vibrio parahaemolyticus and related hemolytic vibrios in marine environments of Washington state Appl Microbiol 20:179–186 Barrow, G.I., and D.C Miller 1976 Vibrio parahaemolyticus and seafoods In Microbiology in Agriculture, Fisheries and Food, ed F.A Skinner and J.G Carr, 181–195 New York: Academic Press Krieg, N.R ed 1984 Bergey’s Manual of Systematic Bacteriology Vol Baltimore: Williams & Wilkins Beuchat, L.R 1973 Interacting effects of pH, temperature, and salt concentration on growth and survival of Vibrio parahaemolyticus Appl Microbiol 25:844–846 Beuchat, L.R 1974 Combined effects of water activity, solute, and temperature on the growth of Vibrio parahaemolyticus Appl Microbiol 27:1075–1080 10 Beuchat, L.R., and R.E Worthington 1976 Relationships between heat resistance and phospholipid fatty acid composition of Vibrio parahaemolyticus Appl Environ Microbiol 31:389–394 11 Beumer, R.R., J.J.M Cruysen, and I.R.K Birtantie 1988 The occurrence of Campylobacter jejuni in raw cows’ milk J Appl Bacteriol 65:93–96 ... inhibited in 21% oxygen Carbon dioxide (about 10%) is required for good growth When C jejuni was inoculated into vacuum-packaged processed turkey meat, cell numbers decreased, but some remained viable... noted that 3–14% of diarrheal patients in developed countries yield stool specimens that contain C jejuni.15 670 Modern Food Microbiology Peak isolations from individually caged hens occurred... It is assumed that the organisms noted were contracted from foods even though this connection is not demonstrated In the case of Campylobacter, it is assumed that about 90% are of food origin