Vol. 29, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2606-2608
0095-1137/91/112606-03$02.00/0 Copyright © 1991, American Society for Microbiology
Feeding Trials of Listeria monocytogenes with a Nonhuman Primate Model J. M. FARBER,* E. DALEY, F. COATES, N. BEAUSOLEIL, AND J. FOURNIER Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario KIA OL2, Canada Received 15 May 1991/Accepted 5 August 1991
One of the major unanswered questions regarding the presence of Listeria monocytogenes in foods is how cells must be ingested in order to cause illness. To answer this question, studies were undertaken by using Macaca fascicularis (cynomolgus monkey) as an animal model. Healthy nonhuman primates were dosed with various concentrations of L. monocytogenes suspended in sterile whole milk. Final concentrations of 105, 107, and 109 total cells of the organism were used; a control was also included. Blood samples, as well as fecal and nasal specimens, were taken at various time intervals. Only animals that received 109 cells of L. monocytogenes became noticeably ill, with symptoms of septicemia, irritability, loss of appetite, and occasional diarrhea. Monkeys that received 107 and 109 cells shed L. monocytogenes in the feces for approximately 21 days. In monkeys that received the dose of 109 cells, severe lymphopenia and neutrophilia occurred within 48 h. In a separate trial, monkeys received Maalox to reduce the gastric acidity of the stomach. However, no substantial differences were observed between Maalox-treated and control monkeys. many
(Difco) with 0.6% yeast extract (TSB-YE). Tubes were then incubated at 30°C for 24 h, the suspension was centrifuged at 2,000 x g for 30 min, and the pellet was resuspended in
Listeria monocytogenes is a foodborne pathogen which has caused many problems for both food industries and regulatory agencies worldwide. The number of L. monocytogenes cells required to cause disease in both normal and debilitated individuals is unknown. This has created many problems from a regulatory point of view, because in some countries (the United States), it has been necessary to recall all foods containing the organism, regardless of the number of cells present. Although there have been a number of feeding trials with L. monocytogenes by using animals such as mice (5, 17), sheep (15), rabbits (6, 14), and goats (13) to determine their suitability as animals models, little work has been done by using the monkey as a model. Thus, studies were undertaken to evaluate the use of the cynomolgus monkey as an animal model and approximate the minimum infectious dose of L. monocytogenes for healthy humans. In each experiment, four or five adult female Macaca fascicularis (cynomolgus monkeys) weighing 3 to 5 kg were used. Each animal, fed ad libitum, was housed in a separate cage, with all cages being kept in a specially designed Labsafe facility (Canadian Cabinets, Nepean, Ontario, Canada). Animals were not retested once they were orally challenged with the test organism, except where indicated otherwise. Serum samples were obtained from the monkeys prior to dosing, to ensure that monkeys with antibody titers against L. monocytogenes greater than 1:64 would not be used in any trials. Organisms used to inoculate monkeys included L. monocytogenes Scott A (serotype 4b) and F6861 (serotype 4b), an isolate from Jalisco cheese. Before use, organisms were passaged once in mice and removed from the spleen or liver by direct plating onto tryptose agar (TA; Difco Laboratories, Detroit, Mich.), LPM agar (Difco), or Oxfôrd agar (Oxoid Inc., Nepean, Ontario, Canada). From the plates, single colonies were transferred into 5 ml of tryptic soy broth *
sterile whole milk. Dilutions in milk were subsequently made to give total final cell concentrations of 109, 107, and 105 cells. The inoculum was administered into the stomachs of the monkeys in a total volume of 10 ml of milk with the aid of a nasogastric tube. Control monkeys included those that received 10 ml of sterile milk and/or no treatment at all. In experiments in which we looked at the effects of reduced gastric acidity on the outcome of infection, 10 ml of Maalox Plus was fed to the monkeys (nasogastric tube) 1 h prior to dosing. AUl experiments were performed in duplicate. For the first 5 days, nasal swabs, blood samples, body temperature, the general appearance of the animal, and food consumption patterns were recorded. Fecal specimens were taken every day for 5 days and then approximately every second day for up to 30 days. Complete hematological profiles were performed on a Coulter counter (S+IV; Coulter Counter, Burlington, Ontario, Canada). The differentials were obtained by microscopic evaluation at x400 power. The fecal and nasal specimens were analyzed for L. monocytogenes by the method of the U.S. Department of Agriculture (10, 11), except that University of Vermont (UVM) enrichment broths were incubated for 7 days, with 0.1 ml being transferred into Fraser broth at days 1 and 7. In addition, LPM and Oxford media were used instead of modified Oxford medium. Confirmation of L. monocytogenes was done as described previously (4). L. monocytogenes Scott A or F6861 was grown at 25°C for 48 h in 50 ml of TSB-YE containing 1% glucose. Formaldehyde solution (final concentration, 0.5% in saline) was then added to the flasks, which were then incubated for an additional 24 h at 25°C. TA plates were streaked from the broth to check for sterility. The suspension was then centrifuged (11,000 x g, 20 min), the cells were washed twice with 0.5% formaldehyde, and the pellet was resuspended in 5 ml of 0.1 M phosphate-buffered saline solution (PBS; pH 7.0).
Corresponding author. 2606
VOL. 29,
1991
NOTES
TABLE 1. Fecal shedding pattern from monkeys orally dosed with L. monocytogenes Time (days)
Control
1 2 3 4 5 7 9
-
-
Total dose of L. monocytogenes F6861' i05 iO9b 107 109
+ +
-
+ + + + +
+ +
+ +
+
+
+ + +
+ + +
+ +
100-
i05
60-
El
+ 40-
-
20-
-
-
-
il
-
-
13 16 18 21 23 25 28 30
-
-+
-
-
+
+
-
-
+
+
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
a _, implies that no L. monocytogenes was isolated; +, implies that L. monocytogenes was isolated; control monkeys received 10 ml of sterile milk. b The same animal was dosed for the second time with 109 cells of strain F6861. c Time zero was the day of dosing.
The antigen was then frozen at -70°C in small vials until it was needed. Tests were done in small disposable glass tubes. Briefly, 0.5 ml of PBS buffer and 0.5 ml of serum were placed into the tubes, and a series of doubling dilutions was performed. The test antigen (0.5 ml), either undiluted or diluted 1:11 in PBS buffer, was added to each tube. All tubes were then placed in a water bath at 60°C for 2 h. The presence of agglutination at the bottom of the tubes was recorded visually 1 h after removal from the water bath and after overnight incubation at 4°C. Controls without serum were included to ensure that agglutination and sedimentation were clearly distinguishable. Results obtained with both strains of L. monocytogenes were similar, and thus data are given only for L. monocytogenes F6861. For the most part, symptoms were observed only in those monkeys that received the largest dose (109 cells) of L. monocytogenes. Low-grade fever (normal body temperature, 37.5 to 38.0°C), septicemia (L. monocytogenes was isolated from the blood in three of the six trials), loss of appetite, irritability and occasional diarrhea were observed. Fever peaked on the second day after dosing. The highest recorded temperature for any animal was 38.9°C. None of the animals was treated with antibiotics at any time. Nasal specimens were almost consistently negative throughout all experiments, with the exception of the isolation of L. monocytogenes on one or two occasions (data not shown). A typical fecal shedding pattern is given in Table 1. Generally, only those animals that received a dose of 107 cells or greater were consistent shedders of the organism. Shedding in these animals usually lasted for 3 weeks. Only limited shedding occurred in those animals that received a dose of 105 L. monocytogenes. Interestingly, the monkey that received the 109 challenge dose of strain F6861 for a second time (about 8 weeks later) did not shed the organism for as long a time period as after receiving the initial dose of 109 cells. Evidence of infection was clearly demonstrated in those animals that received doses of 107 and 109 L. monocytogenes cells; i.e., Iymphopenia and neutrophilia were observed.
109
80-
_ _
-
107
2607
2n
I I IL I
0 2LU4 0 Z 4 TIME (Days) FIG. 1. Lymphocyte (l) and neutrophil (E) profiles for cynomolgus monkeys receiving milk containing 105, 107, or 109 cells of L. O.
0 4
monocytogenes F6861. Time zero was the day of dosing.
This decrease in lymphocytes and the increase in neutrophils were much more evident in those animals that received the largest dose (109) of L. monocytogenes (Fig. 1). Values returned to more or less normal levels by days 3 to 4 postinfection. The percent normal range of lymphocytes and neutrophils in cynomolgus monkeys would be approximately 50 to 70% and 20 to 45%, respectively (3). Monkeys treated with Maalox exhibited blood results, temperature profiles, and fecal shedding patterns similar to those of control monkeys (data not shown). The pH of gastric aspirates was determined whenever possible, with values in control and Maalox-treated monkeys being approximately 2.4 and 7.3, respectively. Agglutination titers on predosed monkeys ranged from 1:16 to 1:128, with most animals giving a titer of either 1:16 or 1:64. For some of the monkeys that received the 109 challenge dose, titers determined approximately 1 to 2 weeks after dosing were in the range of 1:256 to 1:512. No attempt was made to follow the agglutination titers throughout the course of infection. In this investigation, an attempt was made to establish a minimum infectious dose for normal healthy individuals by using the cynomolgus monkey as an animal model. Listeriosis has previously been reported both in pregnant and nonpregnant adult nonhuman primates (7, 19). In addition, a recent outbreak of listeriosis in 11 macaques housed outdoors was recently reported (16). This outbreak caused 10 stillbirths and one case of neonatal septicemia. In that outbreak, restriction enzyme analysis pointed to a common source of contamination for the monkeys. The nature and distribution of the lesions observed in the monkeys were typical of those observed in human perinatal listeriosis (16). This study showed that monkeys that received a dose of 109 cells of L. monocytogenes displayed a response indicative of infection, while those that received the 107 dose did not exhibit the signs and symptoms described above, although lymphopenia and neutrophilia were observed (Fig. 1). Results similar to those of the present study were recently seen in goats orally inoculated with 6 x 109 L. monocytogenes (13). Four of five goats developed fever, with the highest temperatures (41.0 to 41.8°C) occurring 1 to 2 days after dosing. Poor appetite was also observed in those animals.
2608
J. CLIN. MICROBIOL.
NOTES
It was interesting to observe that consistent fecal shedding occurred only in those animals that received 107 organisms. The length of shedding in these animals (ca. 21 days) was somewhat similar to that reported by Miettinen et al. (13) for goats that shed L. monocytogenes in the feces for times ranging from 3 to 15 days. The possibility of fecal to oral spread of the organism in humans would therefore be a possibility only in instances in which individuals consumed large numbers of L. monocytogenes. It was also interesting that the monkey that received a dose of 109 cells for a second time did not shed the organism for as long a period oftime and did not develop the signs and symptoms typical of those found in animals that received only one dose of 109 cells. This suggests that the initial exposure to the organism led to the development of antibodies which may have been associated with the more rapid clearance of L. monocytogenes from the gastrointestinal tract and the absence of clinical symptoms. These results, suggesting the possible involvement of humoral immunity in the elimination of the infection, are very similar to those recently reported for experimentally infected goats (13). Our studies with Maalox-treated monkeys did not demonstrate any significant difference between treated and nontreated monkeys, although we observed a neutralization of the gastric acidity in the stomachs of the monkeys. Antacid consumption was considered a risk factor for acquisition of listeriosis in the outbreak which occurred in Boston in 1979 (8). It was theorized that antacids neutralized gastric acidity, possibility allowing L. monocytogenes to survive ingestion (8). Golnazarian et al. (5), working with C57BL/6J mice, also observed no significant differences in the 50% infectious dose between normal or cimetidine (which decreases gastric acid secretion)-treated mice. They felt that because the mice did not have the underlying condition that required treatment with cimetidine, the results should be interpreted with caution (5). The numbers of ingested cells of L. monocytogenes required to cause illness in normal healthy humans has been estimated in few instances. Two sporadic cases of listeriosis involved the consumption of contaminated salted mushrooms (9) and homemade sausage (2), with both foods containing approximately 3 x 106 to 4 x 106 L. monocytogenes per g. However, it was not clear how much of the product the individuals consumed. One well-documented case of sporadic listeriosis occurred in a healthy individual who consumed contaminated soft cheese. It was estimated that this particular female consumed an oral dose of 2.5 x 109 to 4.3 x 109 L. monocytogenes, with symptoms developing less than 24 h later (1). This infection would be similar to the one we observed in the monkeys. However, it is highly likely that many other individuals, both normal and high-risk individuals, consumed this same soft cheese containing high numbers (>105 CFU/g) of L. monocytogenes without apparently developing listeriosis (12). It is possible, however, that very mild upper gastrointestinal symptoms may have developed in some individuals, which would have gone undetected. We have fortuitously observed a case such as this in Canada, where a 59-year-old female developed a very mild case of listeriosis (no fever, septicemia, or meningitis) after consuming soft cheese containing a total of 2.7 x 106 organisms (3). In addition, in a recent listeriosis outbreak in Connecticut which was epidemiologically linked to the consumption of shrimp, the presence of mild disease was observed in the healthy hosts who consumed the contaminated food (18). This study focused on the development of listeriosis in healthy adult monkeys. The next phase of this study will be
to use drugs to suppress the immune system of the animals. It is hoped that by doing this, it will be possible to more
closely simulate experimental conditions which when listeriosis occurs in debilitated individuals.
develop
We thank J. Edgar, M. Sanche, and C. Martineau of the Animal Resources Division for animal and laboratory support.
REFERENCES 1. Azadian, B. S., G. T. Finnerty, and A. D. Pearson. 1989. Cheese-borne Listeria meningitis in immunocompetent patient. Lancet i:322-323.
2. Cantoni, C., C. Balzaretti, and M. Valenti. 1989. A case of L. monocytogenes human infection associated with consumption of cooked meat pork product. Arch. Vet. Ital. 40:141-142. 3. Farber, J. M. Unpublished data. 4. Farber, J. M., M. A. Johnston, U. Purvis, and A. Loit. 1987. Surveillance of soft and semi-soft cheeses for the presence of Listeria spp. Int. J. Food Microbiol. 5:157-163. 5. Golnazarian, C. A., C. W. Donnelly, S. J. Pintauro, and D. B. Howard. 1989. Comparison of infectious dose of Listeria monocytogenes F5817 as determined for normal versus compromised C57B1/6J mice. J. Food Prot. 52:696-701. 6. Gray, M. L., C. Singh, and F. Thorp, Jr. 1955. Abortion, stillbirth, early death of young in rabbits by Listeria monocytogenes Il. Oral exposure. Proc. Soc. Exp. Biol. 89:169-175. 7. Heldstab, A., and D. Ruedi. 1982. Listeriosis in an adult female chimpanzee (Pan troglodytes). J. Comp. Pathol. 92:609-612. 8. Ho, J. L., K. N. Shands, G. Friedland, P. Eckind, and D. W. Fraser. 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146:520-524. 9. Junttila, J., and M. Brander. 1989. Listeria monocytogenes septicemia associated with consumption of salted mushrooms. Scand. J. Infect. Dis. 21:339-342. 10. McClain, D., and W. H. Lee. 1988. Development ofUSDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71:660-664. 11. McClain, D., and W. H. Lee. 1989. FSIS method for the isolation and identification of Listeria monocytogenes from processed meat and poultry products. Laboratory Communica-
tion No. 57. Revised 24 May 1989. Food Safety and Inspection Service, U.S. Department of Agriculture, Beltsville, Md. 12. McLauchlin, J., M. H. Greenwood, and P. N. Pini. 1990. The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis. Int. J. Food Microbiol. 10:255-262. 13. Miettinen, A., J. Husu, and J. Tuomi. 1990. Serum antibody response to Listeria monocytogenes; listerial excretion, and clinical characteristics in experimentally infected goats. J. Clin. Microbiol. 28:340-343. 14. Osebold, J. W., and T. Inouye. 1954. Pathogenesis of Listeria monocytogenes infections in natural hosts. I. Rabbit studies. J. Infect. Dis. 95:52-66. 15. Osebold, J. W., and T. Inouye. 1954. Pathogenesis of Listeria monocytogenes infections in natural hosts. Il. Sheep studies. J. Infect. Dis. 95:67-78. 16. Paul-Murphy, J., J. E. Markovits, I. Wesby, and J. A. Roberts. 1990. Listeriosis causing stillbirths and neonatal septicemia in outdoor housed macaques. Lab. Anim. Sci. 40:547. 17. Pine, L., G. B. Malcolm, and B. D. Plikaytis. 1990. Listeria monocytogenes intragastric and intraperitoneal approximate 50% lethal doses for mice are comparable, but death occurs earlier by intragastric feeding. Infect. Immun. 58:2940-2945. 18. Riedo, F. X., R. W. Pinner, M. Tosca, M. L. Cartter, L. M. Graves, M. W. Reeves, B. D. Plikaytis, and C. V. Broome. 1990. A Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 972. 19. Vetési, F., A. Balsai, and F. Kemenes. 1972. Abortion in Gray's
monkey (Cercopithecus mona) associated with Listeria monocytogenes. Acta Microbiol. Acad. Sci. Hung. 19:441-443.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p. 2606-2608
0095-1137/91/112606-03$02.00/0 Copyright © 1991, American Society for Microbiology
Feeding Trials of Listeria monocytogenes with a Nonhuman Primate Model J. M. FARBER,* E. DALEY, F. COATES, N. BEAUSOLEIL, AND J. FOURNIER Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario KIA OL2, Canada Received 15 May 1991/Accepted 5 August 1991
One of the major unanswered questions regarding the presence of Listeria monocytogenes in foods is how cells must be ingested in order to cause illness. To answer this question, studies were undertaken by using Macaca fascicularis (cynomolgus monkey) as an animal model. Healthy nonhuman primates were dosed with various concentrations of L. monocytogenes suspended in sterile whole milk. Final concentrations of 105, 107, and 109 total cells of the organism were used; a control was also included. Blood samples, as well as fecal and nasal specimens, were taken at various time intervals. Only animals that received 109 cells of L. monocytogenes became noticeably ill, with symptoms of septicemia, irritability, loss of appetite, and occasional diarrhea. Monkeys that received 107 and 109 cells shed L. monocytogenes in the feces for approximately 21 days. In monkeys that received the dose of 109 cells, severe lymphopenia and neutrophilia occurred within 48 h. In a separate trial, monkeys received Maalox to reduce the gastric acidity of the stomach. However, no substantial differences were observed between Maalox-treated and control monkeys. many
(Difco) with 0.6% yeast extract (TSB-YE). Tubes were then incubated at 30°C for 24 h, the suspension was centrifuged at 2,000 x g for 30 min, and the pellet was resuspended in
Listeria monocytogenes is a foodborne pathogen which has caused many problems for both food industries and regulatory agencies worldwide. The number of L. monocytogenes cells required to cause disease in both normal and debilitated individuals is unknown. This has created many problems from a regulatory point of view, because in some countries (the United States), it has been necessary to recall all foods containing the organism, regardless of the number of cells present. Although there have been a number of feeding trials with L. monocytogenes by using animals such as mice (5, 17), sheep (15), rabbits (6, 14), and goats (13) to determine their suitability as animals models, little work has been done by using the monkey as a model. Thus, studies were undertaken to evaluate the use of the cynomolgus monkey as an animal model and approximate the minimum infectious dose of L. monocytogenes for healthy humans. In each experiment, four or five adult female Macaca fascicularis (cynomolgus monkeys) weighing 3 to 5 kg were used. Each animal, fed ad libitum, was housed in a separate cage, with all cages being kept in a specially designed Labsafe facility (Canadian Cabinets, Nepean, Ontario, Canada). Animals were not retested once they were orally challenged with the test organism, except where indicated otherwise. Serum samples were obtained from the monkeys prior to dosing, to ensure that monkeys with antibody titers against L. monocytogenes greater than 1:64 would not be used in any trials. Organisms used to inoculate monkeys included L. monocytogenes Scott A (serotype 4b) and F6861 (serotype 4b), an isolate from Jalisco cheese. Before use, organisms were passaged once in mice and removed from the spleen or liver by direct plating onto tryptose agar (TA; Difco Laboratories, Detroit, Mich.), LPM agar (Difco), or Oxfôrd agar (Oxoid Inc., Nepean, Ontario, Canada). From the plates, single colonies were transferred into 5 ml of tryptic soy broth *
sterile whole milk. Dilutions in milk were subsequently made to give total final cell concentrations of 109, 107, and 105 cells. The inoculum was administered into the stomachs of the monkeys in a total volume of 10 ml of milk with the aid of a nasogastric tube. Control monkeys included those that received 10 ml of sterile milk and/or no treatment at all. In experiments in which we looked at the effects of reduced gastric acidity on the outcome of infection, 10 ml of Maalox Plus was fed to the monkeys (nasogastric tube) 1 h prior to dosing. AUl experiments were performed in duplicate. For the first 5 days, nasal swabs, blood samples, body temperature, the general appearance of the animal, and food consumption patterns were recorded. Fecal specimens were taken every day for 5 days and then approximately every second day for up to 30 days. Complete hematological profiles were performed on a Coulter counter (S+IV; Coulter Counter, Burlington, Ontario, Canada). The differentials were obtained by microscopic evaluation at x400 power. The fecal and nasal specimens were analyzed for L. monocytogenes by the method of the U.S. Department of Agriculture (10, 11), except that University of Vermont (UVM) enrichment broths were incubated for 7 days, with 0.1 ml being transferred into Fraser broth at days 1 and 7. In addition, LPM and Oxford media were used instead of modified Oxford medium. Confirmation of L. monocytogenes was done as described previously (4). L. monocytogenes Scott A or F6861 was grown at 25°C for 48 h in 50 ml of TSB-YE containing 1% glucose. Formaldehyde solution (final concentration, 0.5% in saline) was then added to the flasks, which were then incubated for an additional 24 h at 25°C. TA plates were streaked from the broth to check for sterility. The suspension was then centrifuged (11,000 x g, 20 min), the cells were washed twice with 0.5% formaldehyde, and the pellet was resuspended in 5 ml of 0.1 M phosphate-buffered saline solution (PBS; pH 7.0).
Corresponding author. 2606
VOL. 29,
1991
NOTES
TABLE 1. Fecal shedding pattern from monkeys orally dosed with L. monocytogenes Time (days)
Control
1 2 3 4 5 7 9
-
-
Total dose of L. monocytogenes F6861' i05 iO9b 107 109
+ +
-
+ + + + +
+ +
+ +
+
+
+ + +
+ + +
+ +
100-
i05
60-
El
+ 40-
-
20-
-
-
-
il
-
-
13 16 18 21 23 25 28 30
-
-+
-
-
+
+
-
-
+
+
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
a _, implies that no L. monocytogenes was isolated; +, implies that L. monocytogenes was isolated; control monkeys received 10 ml of sterile milk. b The same animal was dosed for the second time with 109 cells of strain F6861. c Time zero was the day of dosing.
The antigen was then frozen at -70°C in small vials until it was needed. Tests were done in small disposable glass tubes. Briefly, 0.5 ml of PBS buffer and 0.5 ml of serum were placed into the tubes, and a series of doubling dilutions was performed. The test antigen (0.5 ml), either undiluted or diluted 1:11 in PBS buffer, was added to each tube. All tubes were then placed in a water bath at 60°C for 2 h. The presence of agglutination at the bottom of the tubes was recorded visually 1 h after removal from the water bath and after overnight incubation at 4°C. Controls without serum were included to ensure that agglutination and sedimentation were clearly distinguishable. Results obtained with both strains of L. monocytogenes were similar, and thus data are given only for L. monocytogenes F6861. For the most part, symptoms were observed only in those monkeys that received the largest dose (109 cells) of L. monocytogenes. Low-grade fever (normal body temperature, 37.5 to 38.0°C), septicemia (L. monocytogenes was isolated from the blood in three of the six trials), loss of appetite, irritability and occasional diarrhea were observed. Fever peaked on the second day after dosing. The highest recorded temperature for any animal was 38.9°C. None of the animals was treated with antibiotics at any time. Nasal specimens were almost consistently negative throughout all experiments, with the exception of the isolation of L. monocytogenes on one or two occasions (data not shown). A typical fecal shedding pattern is given in Table 1. Generally, only those animals that received a dose of 107 cells or greater were consistent shedders of the organism. Shedding in these animals usually lasted for 3 weeks. Only limited shedding occurred in those animals that received a dose of 105 L. monocytogenes. Interestingly, the monkey that received the 109 challenge dose of strain F6861 for a second time (about 8 weeks later) did not shed the organism for as long a time period as after receiving the initial dose of 109 cells. Evidence of infection was clearly demonstrated in those animals that received doses of 107 and 109 L. monocytogenes cells; i.e., Iymphopenia and neutrophilia were observed.
109
80-
_ _
-
107
2607
2n
I I IL I
0 2LU4 0 Z 4 TIME (Days) FIG. 1. Lymphocyte (l) and neutrophil (E) profiles for cynomolgus monkeys receiving milk containing 105, 107, or 109 cells of L. O.
0 4
monocytogenes F6861. Time zero was the day of dosing.
This decrease in lymphocytes and the increase in neutrophils were much more evident in those animals that received the largest dose (109) of L. monocytogenes (Fig. 1). Values returned to more or less normal levels by days 3 to 4 postinfection. The percent normal range of lymphocytes and neutrophils in cynomolgus monkeys would be approximately 50 to 70% and 20 to 45%, respectively (3). Monkeys treated with Maalox exhibited blood results, temperature profiles, and fecal shedding patterns similar to those of control monkeys (data not shown). The pH of gastric aspirates was determined whenever possible, with values in control and Maalox-treated monkeys being approximately 2.4 and 7.3, respectively. Agglutination titers on predosed monkeys ranged from 1:16 to 1:128, with most animals giving a titer of either 1:16 or 1:64. For some of the monkeys that received the 109 challenge dose, titers determined approximately 1 to 2 weeks after dosing were in the range of 1:256 to 1:512. No attempt was made to follow the agglutination titers throughout the course of infection. In this investigation, an attempt was made to establish a minimum infectious dose for normal healthy individuals by using the cynomolgus monkey as an animal model. Listeriosis has previously been reported both in pregnant and nonpregnant adult nonhuman primates (7, 19). In addition, a recent outbreak of listeriosis in 11 macaques housed outdoors was recently reported (16). This outbreak caused 10 stillbirths and one case of neonatal septicemia. In that outbreak, restriction enzyme analysis pointed to a common source of contamination for the monkeys. The nature and distribution of the lesions observed in the monkeys were typical of those observed in human perinatal listeriosis (16). This study showed that monkeys that received a dose of 109 cells of L. monocytogenes displayed a response indicative of infection, while those that received the 107 dose did not exhibit the signs and symptoms described above, although lymphopenia and neutrophilia were observed (Fig. 1). Results similar to those of the present study were recently seen in goats orally inoculated with 6 x 109 L. monocytogenes (13). Four of five goats developed fever, with the highest temperatures (41.0 to 41.8°C) occurring 1 to 2 days after dosing. Poor appetite was also observed in those animals.
2608
J. CLIN. MICROBIOL.
NOTES
It was interesting to observe that consistent fecal shedding occurred only in those animals that received 107 organisms. The length of shedding in these animals (ca. 21 days) was somewhat similar to that reported by Miettinen et al. (13) for goats that shed L. monocytogenes in the feces for times ranging from 3 to 15 days. The possibility of fecal to oral spread of the organism in humans would therefore be a possibility only in instances in which individuals consumed large numbers of L. monocytogenes. It was also interesting that the monkey that received a dose of 109 cells for a second time did not shed the organism for as long a period oftime and did not develop the signs and symptoms typical of those found in animals that received only one dose of 109 cells. This suggests that the initial exposure to the organism led to the development of antibodies which may have been associated with the more rapid clearance of L. monocytogenes from the gastrointestinal tract and the absence of clinical symptoms. These results, suggesting the possible involvement of humoral immunity in the elimination of the infection, are very similar to those recently reported for experimentally infected goats (13). Our studies with Maalox-treated monkeys did not demonstrate any significant difference between treated and nontreated monkeys, although we observed a neutralization of the gastric acidity in the stomachs of the monkeys. Antacid consumption was considered a risk factor for acquisition of listeriosis in the outbreak which occurred in Boston in 1979 (8). It was theorized that antacids neutralized gastric acidity, possibility allowing L. monocytogenes to survive ingestion (8). Golnazarian et al. (5), working with C57BL/6J mice, also observed no significant differences in the 50% infectious dose between normal or cimetidine (which decreases gastric acid secretion)-treated mice. They felt that because the mice did not have the underlying condition that required treatment with cimetidine, the results should be interpreted with caution (5). The numbers of ingested cells of L. monocytogenes required to cause illness in normal healthy humans has been estimated in few instances. Two sporadic cases of listeriosis involved the consumption of contaminated salted mushrooms (9) and homemade sausage (2), with both foods containing approximately 3 x 106 to 4 x 106 L. monocytogenes per g. However, it was not clear how much of the product the individuals consumed. One well-documented case of sporadic listeriosis occurred in a healthy individual who consumed contaminated soft cheese. It was estimated that this particular female consumed an oral dose of 2.5 x 109 to 4.3 x 109 L. monocytogenes, with symptoms developing less than 24 h later (1). This infection would be similar to the one we observed in the monkeys. However, it is highly likely that many other individuals, both normal and high-risk individuals, consumed this same soft cheese containing high numbers (>105 CFU/g) of L. monocytogenes without apparently developing listeriosis (12). It is possible, however, that very mild upper gastrointestinal symptoms may have developed in some individuals, which would have gone undetected. We have fortuitously observed a case such as this in Canada, where a 59-year-old female developed a very mild case of listeriosis (no fever, septicemia, or meningitis) after consuming soft cheese containing a total of 2.7 x 106 organisms (3). In addition, in a recent listeriosis outbreak in Connecticut which was epidemiologically linked to the consumption of shrimp, the presence of mild disease was observed in the healthy hosts who consumed the contaminated food (18). This study focused on the development of listeriosis in healthy adult monkeys. The next phase of this study will be
to use drugs to suppress the immune system of the animals. It is hoped that by doing this, it will be possible to more
closely simulate experimental conditions which when listeriosis occurs in debilitated individuals.
develop
We thank J. Edgar, M. Sanche, and C. Martineau of the Animal Resources Division for animal and laboratory support.
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