606 Modern Food Microbiology that caused a 50% infectious dose (ID50 ) were 3.24–4.55 log cfu for normal mice, 1.91–2.74 for the cortisone-treated, and 2.48 for pregnant mice.50 The ID50 for those administered cimetidine was similar to the normals These investigators found no significant difference between IP and IG administration relative to ID50 Employing neonatal mice (within 24 hours of birth), the LD50 by IP injection of L monocytogenes was 6.3 × 10/cfu, but for 6- to 8-week-old mice, the LD50 was 3.2 × 106 by the same route of administration.17 The neonatal mice were protected against a lethal dose of L monocytogenes when γ -interferon was injected (see below) With 15- to 20-g Swiss mice treated with carrageenan, LD50 was found to range from about to 3,100 cfu.25 When nude mice are challenged with virulent strains of L monocytogenes, chronic infections follow, and for baby mice and macrophage-depleted adult mice, virulent strains are lethal With the adult mouse model, rough strains of L monocytogenes multiplied only weakly, and a weak immunity was induced; baby mice were killed, but nude mice survived.59 In nonfatal infections by virulent strains, the organisms multiply in the spleen, and protection against reinfections results regardless of the serovar used for subsequent challenge.59 Overall, studies with the mouse model confirm the greater susceptibility to L monocytogenes of animals with impaired immune systems than normal animals, as is the case with humans The correspondence of minimal infectious doses for normal adult mice to humans is more difficult It has been suggested that levels of L monocytogenes less than 102 /cfu appear to be inconsequential to healthy hosts.50 From the nine cheeses reported by Gilbert and Pini47 that contained 104 –105 /g of L monocytogenes, no known human illness resulted INCIDENCE AND NATURE OF THE LISTERIOSIS SYNDROMES Incidence Although L monocytogenes may have been described first in 1911 by Hăulphers,67 its unambiguous description was made in 1923 by Murray et al.98 Since that time it has been shown to be a pathogen in over 50 mammals, including humans, in addition to fowls, ticks, fish, and crustaceans The first human case of listeriosis was reported in 1929, and the disease has since been shown to occur sporadically throughout the world L monocytogenes is the etiological agent of about 98% of human and 85% of animal cases.92 At least three human cases have been caused by L ivanovii and one by L seeligeri There were around 60 human cases in the United Kingdom in 1981 but around 140 in 1985, along with a similar increase in animal cases.93 Between 1986 and 1988, human listeriosis increased in England and Wales by 150%, along with a 100% increase in human salmonellosis The overall mortality rate for 558 human cases in the United Kingdom was 46%, with 51 and 44%, respectively, for perinatal and adult cases.93 For the period 1983–1987, 775 cases were reported in Britain, with 219 (28%) deaths, not including abortions When the 44 abortions are added to the deaths, the fatality rate is 34%.53 Prior to 1974, 15 documented cases were seen yearly in western France, but in 1975 and 1976 there were 115 and 54, respectively.16 All but of 145 strains that were serotyped were serotype There were 687 cases in France in 1987.22 In a 9-year period prior to early 1984, Lausanne, Switzerland, experienced a mean of cases of human listeriosis per year, but in a 15-month period in 1983–1984, 25 cases were seen.90 Thirty-eight of 40 strains examined were serovar 4b, and 92% had the same phage type By and large, foodborne outbreaks of human listeriosis seemed to have waned over the past several years with a few exceptions, as may be noted from Table 25–5 In the early to mid-1990s, the estimated Foodborne Listeriosis 607 number of cases/million persons in several countries were as follows: Australia (1992) Canada Denmark United Kingdom United States 2–4 4–5 2–3 ∼4 The estimated number of cases in the United States for 1993 was 1092 with 248 deaths Not all cases are of direct food origin, as other sources have been documented From a risk assessment study, a person on average is exposed 3.8 times via food to 5.0 log10 organisms and 0.8 times to >106 log10 organisms/year with about five to seven cases of listeriosis per year.101 After considering other factors such as mouse infective doses, the investigators concluded that listeriosis is a rare disease in humans despite frequent exposure to the causative organism Source of Pathogens With the incidence of human foodborne listeriosis being so low and sporadic, the source of the causative strains of L monocytogenes is of great interest Although the outbreaks traced to dairy products may be presumed to result from the shedding of virulent strains into milk, this is not always confirmed In a study of 1,123 raw milk samples from the 27 farms that supplied milk to the incriminated cheese plant in California in 1985, Donnelly et al.28 were unable to recover the responsible 4b serovar A serotype was isolated from 16 string samples from one control farm In a review of the human cases through most of 1986, Hird57 concluded that whereas the evidence was not conclusive in all cases, it nevertheless supported zoonotic transmission to some degree (zoonosis: disease transmissible under natural conditions from vertebrate animals to humans) Hird believes the healthy animal carrier is an important source of the organism, along with clinical listeriosis in livestock, but the relative degree to which each contributes to foodborne cases is uncertain L monocytogenes was found to be shed in milk from the left forequarter of a mastitic cow, but milk from the other quarters was uninfected.48 About 10% of healthy cattle tested in The Netherlands were positive for L monocytogenes, and about 5% of human fecal samples from slaughterhouse workers in Denmark contained the organism.68 The carriage rate for healthy humans seemed to be about the same regardless of their work position within food processing plants.68 Over an 18-month period in the United Kingdom, 32 of 5,000 (0.6%) fecal samples were positive.75 Cross-infection with L monocytogenes from congenitally infected newborn infants to apparently healthy neonates in hospitals has been shown to occur.94 Thus, although the organism is known to be fairly common in environmental specimens, it also exists in healthy humans at rates from less than 1% to around 15% The relative importance of environmental, animal, and human sources to foodborne episodes awaits further study Among ready-to-eat (RTE) foods, meat and poultry products are the leading vehicles for human listeriosis In its annual survey of RTE meat and poultry products, the Food Safety and Inspection Service (FSIS) of the U.S Department of Agriculture found the following percent of these products to contain L monocytogenes during the years noted: 1995—3.02 1996—2.91 1997—2.25 1998—2.54 1999—1.91 2000—1.45 2001—1.32 2002—1.03 2003—0.75 (January–September) 608 Modern Food Microbiology Table 25–5 Some of the Suspected and Proven Foodborne Listeriosis Outbreaks and Cases Year 1953 1959 1960–1961 1966 1979 1980 1981 1983 1983–1987 1985 1986–1987 1987–1989 1987 1988 1988 1988 1988 1989 1988 1989 1989 1989 1990 1990 1990 1991 1992 1992 1992 1993 1994 1994 1995 1998–1999 1999–2000 2000–2001 2002 ∗ Suspected † Epidemiologically Source Cases/Deaths Location Raw milk Fresh meat/poultry∗ Various/unknown Milk/products Vegetables/milk?† Shellfish Cole slaw Pasteurized milk† Vacherin Mont D’Or Mexican-style cheese Vegetables?† Pat ˆ e´ Soft cheese Goats’ milk cheese Cooked-chld-chick Cooked-chld-chick Turkey franks Pork sausage Alfalfa tablets Salted mushrooms Shrimp Pork sausage Raw milk Pork sausage Pat ˆ e´ Smoked mussels Smoked mussels Goat meat (from Calif.) Pork tongue in jelly Pork rillettes Chocolate milk Pickled olives Brie cheese Wieners Pork tongue in jelly Homemade Mexican-style cheese Deli turkey meat 2/1 4/2 81/? 279/109 23/3 22/6 41/18 49/14 122/34 142/48 36/16 366/63 1 1 1 9/1 1 11/6 3/0 4/2 279/85 39/0 52/0 17/0 ca 101/ca 21 26/7 12/0 46/7 Germany Sweden Germany Germany Boston New Zealand Canada Boston Switzerland California Philadelphia United Kingdom United Kingdom United Kingdom United Kingdom United Kingdom Oklahoma Italy Canada Finland United States (Conn.) Italy Vermont Italy Australia Australia New Zealand Canada France France USA Italy France United States France United States 10 USA states linked; organisms not found In an extensive review of this organism in the general processing environment, Tompkin124 noted that foodborne listeriosis accounted for only ca 0.02% of all foodborne illnesses in the United States On the other hand, this disease accounted for ca 28% of all deaths from foodborne illnesses Foods that have been involved in human cases typically contain >1,000 cfu/g or ml; and outbreak strains typically have become established in the processing environment resulting in contamination of multiple lots of foods.124 Foodborne Listeriosis 609 In spite of the high fatality rate associated with foodborne listeriosis, overall the disease is relatively rare Not all L monocytogenes isolates are equally pathogenic, and just what makes a foodborne isolate infectious or noninfectious is poorly understood as are the reasons for the predominance in foods of serotypes 1/2a, 1/2b, and 4b.69 Syndromes Listeriosis in humans is not characterized by a unique set of symptoms because the course of the disease depends on the state of the host Non-pregnant healthy individuals who are not immunosuppressed are highly resistant to infection by L monocytogenes, and there is little evidence that such individuals ever contract clinical listeriosis However, the following conditions are known to predispose to adult listeriosis and to be significant in mortality rate: neoplasm, AIDS, alcoholism, diabetes (type 1, in particular), cardiovascular disease, renal transplant, and corticosteroid therapy When susceptible adults contract the disease, meningitis and sepsis are the most commonly recognized symptoms Of 641 human cases, 73% of victims had meningitis, meningoencephalitis, or encephalitis Cervical and generalized lymphadenopathy are associated with the adult syndrome, and thus the disease may resemble infectious mononucleosis Cerebrospinal fluid initially contains granulocytes, but in later states, monocytes predominate Pregnant females who contract the disease (and their fetuses are often congenitally infected) may not present any symptoms, but when they do, they are typically mild and influenzalike Abortion, premature birth, or stillbirth is often the consequence of listeriosis in pregnant females When a newborn is infected at the time of delivery, listeriosis symptoms typically are those of meningitis, and they typically begin 1–4 weeks after birth, although a 4-day incubation has been recorded The usual incubation time in adults ranges from to several weeks Among the 20 case patients studied from the cluster of cases in the Boston episode, 18 had bacteremia, developed meningitis, and 13 complained of vomiting, abdominal pain, and diarrhea 72 hours before onset of symptoms The control of L monocytogenes in the body is affected by T lymphocytes and activated macrophages, and thus any condition that adversely affects these cells will exacerbate the course of listeriosis The most effective drugs for treatment are coumermycin, rifampicin, and ampicillin, with the last plus an aminoglycoside antibiotic being the best combination.33 Even with that regimen, antimicrobial therapy for listeriosis is not entirely satisfactory because ill patients and compromised hosts are more difficult than competent hosts RESISTANCE TO LISTERIOSIS Resistance or immunity to intracellular pathogens such as viruses, animal parasites, and L monocytogenes is mediated by T cells, lymphocytes that arise from bone marrow and undergo maturation in the thymus (hence, T for thymus derived) Unlike B cells, which give rise to humoral immunity (circulating antibodies), activated T cells react directly against foreign cells Once a pathogen is inside a host cell, it cannot be reached by circulating antibody, but the presence of the pathogen is signaled by structural changes in the parasitized cell, and T cells are involved in the destruction of this invaded host cell, which is no longer recognized as “self.” Macrophages are important to the actions of T cells, and their need for the destruction of L monocytogenes and certain other intracellular pathogens was shown by Mackeness.87 First, macrophages bind and “present” L monocytogenes cells to T cells in such a way that they are recognized as being foreign When T cells react with the organism, they increase in size and form clones specific for the same organism or antigen These T cells, said to be activated, secrete interleukin-1 (IL-1) As the activated T cells multiply, they differentiate to form various subsets 610 Modern Food Microbiology The most important subsets of T cells for resistance to listeriosis are helper or CD4 (L3T4+ ) and cytolytic (killer) or CD8 (Lyt2+ ).71 The CD4 T cells react with the foreign antigen, after which they produce lymphokines (cytokines): IL-1, IL-2, IL-6, immune or γ -interferon, and others γ -Interferon, whose production is aided also by tumor necrosis factor,22 induces the production of IL-2 receptor expression on monocytes Also, IL-2 may enhance the activation of lymphokine-activated killer cells that can lyse infected macrophages As little as 0.6 µg per mouse of exogenously administered IL-2 has been shown to strengthen mouse resistance to L monocytogenes.54 γ -Interferon activates macrophages and CD8 T cells, and the latter react with L monocytogenes-infected host macrophages—and cause their lysis Both CD4 and CD8 T cells are stimulated by L monocytogenes; they activate macrophages via their production of γ -interferon and contribute to resistance to listeriosis.70 CD8 also secretes γ -interferon when exogenous IL-2 is provided, and both CD4 and CD8 can confer some passive immunity to recipient mice.70 Some of the events that occur in murine hosts following infection with L monocytogenes are presumed to be the same events that occur in humans When macrophages engulf L monocytogenes, a factor-increasing monocytopoiesis (FIM) is secreted by the macrophages at the infection site FIM is transported to the bone marrow, where it stimulates the production of more macrophages Only viable L monocytogenes cells can induce the T cell response and immunity to histeriosis Because LLO is the virulence factor of L monocytogenes that elicits the T cell response, this heat-labile protein is destroyed when cells are heat killed The CD8 T cell subset appears to be the major T cell component responsible for antilisterial immunity, for it acts by eliciting lymphokine production by macrophages; passive immunity to L monocytogenes can be achieved by transfer of the CD8 T cell subset.4 The T cell response does not occur even when mice are injected with both killed cells and recombinant IL-1a.60 Neither avirulent nor killed cells induce IL-1 in vitro; viable L monocytogenes cells do,96 indicating a critical role for IL-1 and γ -interferon in the initiation of the in vivo response, for it has been shown that simultaneously administered IL-1a and γ -interferon increased resistance to L monocytogenes in mice better than either alone.74 The combination was not synergistic, only additive It appears that the primary role of γ -interferon is to elicit lymphokine production rather than acting directly, and it is known to increase the production of IL-1.17 γ -Interferon is detectable in the blood stream and spleen of mice only during the first days after infection.99 Infection of mice by L monocytogenes leads to an increase in IL-6, which is produced by nonlymphocyte cells.80 Mice that are deficient in IL-6 have increased susceptibility to listeriosis.23 IL-6 appears to act by stimulating the production of neutrophils.23 This synopsis of murine resistance to L monocytogenes reveals some of the multifunctional roles of the lymphokines in T cell immunity (due to different E-cadherins needed for InlA receptors); and the apparent critical importance of LLO as the primary virulence factor of this organism What makes immunocompromised hosts more susceptible to listeriosis is the dampening effect that immunosuppressive agents have on the T cell system Possible therapy is suggested by the specific roles that some of the lymphokines play, but whether the effects are similar in humans is unclear PERSISTENCE OF L MONOCYTOGENES IN FOODS Because it can grow over the temperature range of about 1–45◦ C and the pH range of 4.1 to around 9.6, L monocytogenes may be expected to survive in foods for long periods of time, and this has been confirmed Strains Scott A and V7, inoculated at levels of 104 –105 /g, survived in cottage cheese for up to 28 days when held at 3◦ C.111 When these two strains, with two others, were inoculated into a camembert cheese formulated with levels of 104 –105 , growth occurred during the first 18 days of ... Tompkin124 noted that foodborne listeriosis accounted for only ca 0.02% of all foodborne illnesses in the United States On the other hand, this disease accounted for ca 28% of all deaths from foodborne... 12/0 46/7 Germany Sweden Germany Germany Boston New Zealand Canada Boston Switzerland California Philadelphia United Kingdom United Kingdom United Kingdom United Kingdom United Kingdom Oklahoma... milk cheese Cooked-chld-chick Cooked-chld-chick Turkey franks Pork sausage Alfalfa tablets Salted mushrooms Shrimp Pork sausage Raw milk Pork sausage Pat ˆ e´ Smoked mussels Smoked mussels Goat