Vol. 12, No. 6 Printed in U.S.A.
INFECTION AND IMMUNITY, Dec. 1975, p. 1319-1324 Copyright©) 1975 American Society for Microbiology
Increased Susceptibility to Bacterial Infection as a Sequela of Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J. E. THIGPEN,* R. E. FAITH, E. E. McCONNELL, AND J. A. MOORE National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received for publication 12 August 1975
The effects of subclinical levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) the response of mice to infection with either Salmonella bern or Herpesvirus suis, also known as pseudorabies virus, are reported. TCDD is a contaminant of certain commercially useful chemicals, such as chlorinated phenols or herbicides. It has been shown to cause thymic atrophy and to suppress cell-mediated immunity in laboratory animals. Sublethal levels of TCDD were used: 0.5, 1, 5, 10, or 20 Mg/kg, given through a gastric tube once weekly for 4 weeks. A significant decrease in weight gain compared with control mice occurred at the 20-,ug dosage. Dose schedules of 1 ,ug or more, followed by salmonella infection, resulted in significant increases in mortality and decreases in the time from infection to death. However, TCDD had no significant effect on mortality in the pseudorabies-infected mice. The most important finding in this study is that extremely low levels of TCDD, which do not produce clinical or pathological change, still have the capacity to affect host defense. on
Various environmental chemicals have been shown to affect immune responses or host resistance to infectious agents (Friend and Trainer [6], Gainer [81, Gainer and Pry [10], Hemphill et al. [11], Jones et al. [14], Koller and Thigpen [191, Koller and Kovacic [18], Koller [171, Verschuuren et al. [27], Vos and van Genderen [30], Vos et al. [291, Vos and Moore [28], Wassermann et al. [32]). These reports generally involve observed defects in antibody response (14, 17, 18, 19, 27, 30, 32) or on cell mediated immunity (28, 29, 30), although several (6, 8, 10, 11) also describe suppressed host resistance to infection. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) can occur as a highly toxic contaminant in the production of some chlorinated phenols or chemicals synthesized from chlorinated phenols such as the herbicide 2,4,5-trichlorophenoxyacetic acid and thus could be unknownly distributed in the environment. TCDD as an environmental contaminant can present health problems, both in man and other animals. It is associated with chloracne in man (16) and chick edema disease in chickens (5). More recently TCDD and 2,4,5-trichlorophenol were implicated as the probable cause of toxicity in a group of horses in eastern Missouri. In addition to the horses, insects, birds, and rodents were poisoned, and there were several cases of human illness attributed to TCDD in this incident (3). TCDD has been observed to cause profound thymic atrophy and suppress cell-mediated im-
munity in rats, mice, and guina pigs (28, 29). It is not known whether TCDD-exposed animals are more susceptible to infectious agents. These studies were designed to determine the effects of subclinical levels of TCDD on the response of mice subsequently infected with either Salmonella bern or pseudorabies virus (PRV). The criteria used to measure these effects were clinical appearance, weight gains, mortality rates, time from infection to death, and pathological
examination. MATERIALS AND METHODS Animal. Four-week-old male C57BL/6Jfh (J67) specific-pathogen-free mice were purchased from
Jackson Laboratories, Bar Harbor, Me. The mice were caged individually under filter tops (Envirogard, Lab Products, Garfield, N.J.) following a rigid sanitary regimen and given food (Sterilizable LabBlox, Allied Mills, Inc., Chicago, Ill.) and water ad libitum. TCDD. TCDD, greater than 99% purity (lot 851144-II), was a gift from Dow Chemical Company, Midland, Mich. It was dissolved in reagent-grade acetone and subsequently diluted with at least 6 parts of corn oil. The exact volume of corn oil was adjusted to provide each mouse with a dose volume of approximately 0.2 ml. Infectious agents. The bacterium used was a S. bern isolate from a naturally infected opossum. This organism was grown overnight at 35 to 37 C on Trypticase soy agar containing 5% sheep blood. Bacterial dilutions were prepared in sterile physiological saline, and the number of bacteria was quanti-
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INFECT. IMMUN.
THIGPEN ET AL.
tated turbidimetrically with a Coleman model 6/20 spectrophotometer at 540 ,um. In addition, the number of total viable bacteria in all dilutions used for animal inoculation was confirmed by conventional total plate count procedures. The viral agent used was Herpesvirus suis (PRV) (9). PRV was produced in 75-cm2 Falcon flasks containing RK-13 monolayers. After a 4-plus cytopathogenic effect was observed, the medium from each flask was centrifuged at 800 x g to remove large cellular debris. The supernatants from each flask were pooled, thoroughly mixed, aliquoted in 2-ml vials, and stored at -79 C until used. At the time of inoculation the original suspension contained approximately 5.0 x 10' tissue culture infective doses/ ml. The pooled viral suspension was culture negative for mycoplasma and bacteria. Preliminary study. Lethal dose response curves were established for S. bern and PRV to determine challenge doses (0.2 ml intraperitoneally [i.p.]) that would produce 20% mortality in 8-week-old normal mice. All mice were observed for 14 days. Dead mice were examined by both microbiological and histopathological procedures to verify infection. Experimental design. Two different experiments were performed. In each experiment 4-week-old mice were randomly divided into four groups. Each mouse was weighed and dosed by gavage once each week for 4 consecutive weeks. Mice were dosed with either TCDD in acetone-corn oil or with acetone-
nation were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 ,um, and routinely stained with hematoxylin and eosin. The criteria used to compare the effects of sublethal levels of TCDD in mice subsequently infected with S. bern or PRV were clinical appearance, weight gains, mortality rates, time from infection to death, and pathological examination. Statistical analyses. For the mortality data, dose response trends were analyzed by the Bartholomew test (2), and pair-wise comparisons were made by the Fisher exact test for 2 x 2 tables (12). For the other variables, the significance of dose-response trends was determined by the Jonckheere test (13), and treatment-control differences were evaluated by the two-sided Mann-Whitney ,u tests (12).
RESULTS Effects of TCDD on weight gain, mortality rate, and time from infection to death. There was essentially no difference in mean group body weights prior to TCDD dosing. By week 4, the high-dose group (20 gg of TCDD per kg) gained significantly (P < 0.01) less weight than did the controls. The 10 jig of TCDD per kg dose group also gained less weight; all other TCDD group body weights were comparable to the group which received acetone-corn oil (Table 1). At a given level of TCDD there were no significorn oil. cant differences in the week 4 weights among In the first experiment, each mouse received the mice assigned to the bacteria, virus, or either 0, 5, 10, or 20 ,ug of TCDD/kg per week. Two control groups. There was little association bedays after the last dose of TCDD, when the mice tween body weight at the time of infection and were 8 weeks old, each group was randomly divided into three subgroups. Mice in one subgroup were subsequent survival. Mortality rates in the bacteria and virus coninjected i.p. with 10' viable S. bern organisms (LD20114 dose), the second subgroup received 104 TCID trol groups that had not received TCDD were as of PRV (LD2014 dose), and the third subgroup (con- anticipated based on the LD2014 preliminary trol) was injected with saline only. (LD204,, is the study (Table 2). The groups of mice exposed to number of bacterial or viral agents required to kill 1, 5, 10, or 20 ,ug of TCDD per kg and given S. approximately 20% of the mice inoculated within a bern showed a significant (P < 0.05 to 0.01) 14-day observation period.) The second experiment was conducted in a man- increase in mortality rates. The mortality rate identical to the first except that each mouse received either 0, 0.5, 1, or 5 jig of TCDD/kg per week and the PRV subgroup was eliminated. Both experiments were performed as blind studies, and the order of inoculation was randomized. Neither the bacterium nor the virus appeared to lose or gain potency during the time of administration. The level of mortality seen in the final 25% of the animals inoculated was not statistically different from that observed in the first 25% or the middle 50%. Therefore, the order of inoculation was virtually irrelevant to subsequent survival. After bacterial and viral challenge, the animals were observed at least twice daily for 14 days. Body weights were recorded at death or at termination of the study. Necropsies were performed on at least three mice from each subgroup, and selected tissues were obtained for histopathological and microbiological examination. Mice from subgroups with less than three deaths were sacrificed and necropsied on day 14. The tissues selected for histopathological exami-
ner
TABLE 1. Effect of TCDD on weight gain of mice over a 3-week exposure period Weekly TCDD dosef (g/kg of body wt)
No.
No.
Initial wt (g)b
Wt gain hleg at challenge a
ofe micl()
Expt I 20 10 5 0
Expt II 5 1 0.5 0
60 59 60 60
18.68 18.78 18.41 18.65
± 0.21 ± 0.22
35 33 35 35
20.81 21.08 20.67 21.13
+ + + +
+ 0.22 + 0.19 0.21 0.22 0.24 0.24
3.06 0.20 5.21 + 0.17 5.77 + 0.19 5.65 + 0.17
4.56 4.50 4.06 4.29
+ + + +
0.17 0.17 0.19 0.14
' TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. bMean ± standard error. Cp < 0.01.
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TCCD-DECREASED HOST RESISTANCE TO INFECTION
VOL. 8, 1975
in the 0.5 ug of TCDD per kg dose group was identical to the control group; therefore, the "no effect" level based on the parameters of this study would be somewhere between 0.5 and 1.0 ,ug of TCDD per kg (Table 2; Fig. 1). The mortality rates in the groups that re-
ceived virus were not seen to differ significantly with variations in the dose of TCDD. There was a significant (P < 0.01) dose-related decrease in time from infection to death in the S. bern-challenged mice (Table 3; Fig. 1).
TABLE 2. Comparison of 14-day mortality rates of mice exposed to sublethal levels of TCDD and challenged with either S. bern or PRV Weekly Mortality rates (%) TCDD dosea
(/Ag/kg
of body wt) Expt I 20 10 5 0
Viral
Bacterial challengeb
challengee
95 (19/20)'-f 65 (13/20)° 65 (13/20)9 25 (5/20)
10 (2/20) 0 (0/20) 15 (3/20) 15 (3/20)
Experiment I 80 _ _ __ _
60k
Controld 0 0 0 0
(0/20) (0/19) (0/20) (0/20)
Expt II ND, 0 (0/15) 70 (14/20)9 5 8 (1/13) ND 1 65 (13/20)9 0 (0/15) ND 25 (5/20) 0.5 ND 0 (0/15) 0 25 (5/20) a TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. bBacterial challenge (LD20114) of S. bern via i.p. injection on day 2 after the 4th weekly dose of TCDD. c Viral challenge (LD20114) of PRV via i.p. injection on day 2 after the 4th weekly dose of TCDD. d Control animals were injected i.p. with sterile saline on day 2 after the 4th weekly dose of TCDD. ' The numbers in parentheses indicate the number of mice dying per the total number of mice in the group. fP < 0.01. oP < 0.05. hND, Not done.
*20 ,pg/kg AIO pg/kg @5 pg/kg 0O ug/kg
40
2
20
E
A_ u
>
_
_
-
f1
Experiment I
E
80K
60K
E
1
0 z40-
A 50 .pg/kg ... .I A 1.0 pg/kg 0* 0.5 pg/kg
>
-
-
fK
o000 Ag/kg
/
220 _
0
/ 2
6 8 Time (days)
4
12
10
14
FIG. 1. Cumulative mortality rates and time from infection to death in mice exposed to sublethal levels of TCDD and subsequently infected with S. bern.
TABLE 3. Average time from infection to death (days) Viral challenge'
Bacterial challengeb
Weekly TCDD dosea (jIg/kg of body wt)
No. of
nonsurvivorsd Expt I 20 10 5 0
Time to deathe
19 13 13 5
2.95 0.49f 5.69 0.67 4.54 + 0.849 8.40 1.69
14 13 5 5
2.86 6.08 9.00 7.40
No. of
nonsurvivorsd 2 0 3 3
Time to death"
9.00 + 5.00
6.33 4.67
1.21 0.33
Expt II 5.0 1.0 0.5 0
0.38' 0.92 1.95 1.81
NDh ND ND ND
TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. Bacterial challenge (LD20114) of S. bern via i.p. injection on day 2 after the 4th weekly dose of TCDD. c Viral challenge (LD20,,4) of PRV via i.p. injection on day 2 after the 4th weekly dose of TCDD. d The number of nonsurvivors was the number of animals dying within 14 days after bacterial or viral challenge. e X standard error. a
b
fP < 0.01. 9p < 0.05. h
ND, Not done.
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THIGPEN ET AL.
Pathological observations. Postmortem examination of mice treated with TCDD only revealed dose-dependent lesions in the liver and thymus similar to those described by Vos et al. (31). The thymus from mice in the 20 ,ug/kg group was smaller than normal. Microscopic examination revealed cortical atrophy due to loss of small lymphocytes. Thymic atrophy was much less apparent and often equivocal in the 10 pLg/kg group and was not observed at the lower doses. Interestingly, no lesions were found in the spleen. The livers from the 20 ,tg/kg group were lighter in color than normal and were mottled. This was less evident in the 10 ,ug/kg animals, and no macroscopic lesions were visible in the livers from mice in the 5, 1, or 0.5 ,ug of TCDD per kg dose groups. Histopathological examination of livers from two high-dose groups revealed a nodular surface with variable degrees of hepatocellular cytomegaly and occasional foci of single cell necrosis. A few inflammatory cells (predominantly mononuclear) were found in the areas of necrosis. In addition, there were often intracellular accumulations oflipid in hepatocytes, especially in midzonal areas. Lesions were not observed at lower doses or in other organs.
The lesions described were still apparent in the mice from the two highest dose groups inoculated with virus or bacteria (both survivors and nonsurvivors). In addition, mice in the bacterial groups that died showed macro- and microscopic lesions typical of salmonellosis (1, 26). There was widespread infiltration of polymorphonuclear leukocytes, particularly in visceral organs. Focal necrotic hepatitis resembling microinfarction was attributed to adjacent areas of vasculitis and thrombosis. Lymphoid necrosis, thrombotic vasculitis, and suppuration were also observed in the spleen. Necrotic enteritis, purulent peritonitis, and occasionally suppurative meningitis were also noted. In the virus portion of the experiment, lesions attributable to PRV were not observed in any of the mice examined. PRV in laboratory rodents is known to produce few lesions at the light microscopic level (21).
DISCUSSION The data presented in this study clearly established that mice exposed to four weekly doses of 1, 5, 10, or 20 ,ug of TCDD per kg and subsequently infected with an LD20/14 dose of S. bern have a significantly higher rate of mortality with a shortened incubation period. Furthermore, the mice exposed to 1 or 5 ,ug of TCDD per kg appeared clinically normal, had a normal
INFECT. IMMUN.
rate of weight gain, and showed no histopathological evidence of toxicity. This demonstrates the most important aspect of this study, i.e., that extremely low levels of TCDD which do not produce clinical or pathological change still have the capacity to affect host defense. These findings are significant in that humans may be exposed to environmental contaminants such as TCDD in minute quantities and therefore may be rendered more susceptible to certain types of infectious agents. The fact that TCDD did not increase mortality in mice challenged with PRV suggests a difference in the pathogenesis of these two agents. The reason(s) for this difference is not understood. There are several host defense factors which could account for the increased susceptibility to a facultative intracellular organism such as S. bern. These include: (i) a defect in cell-mediated immunity either through loss of mediating cells or by inhibition of cell function; (ii) a defect on the humoral system; (iii) a defect in one or more of the steps of phagocytosis; and (iv) a reduction or loss of phagocytic cells. Cell-mediated immune reactions as evidenced by delayed hypersensitivity have been shown to play an important role in immunity and recovery from infections with salmonella and other facultative intracellular organisms (4, 15, 20, 22, 23, 25). Delayed hypersensitivity is thought to be mediated through production of lymphokines by specifically activated T cells in response to bacterial antigens (4, 20, 22, 23, 25). TCDD has been shown to suppress delayed hypersensitivity reactions and responses of T cells to activation by the mitogens concanavalin A and phytohemagglutinin (28, 29). These studies did not investigate the influence of TCDD on lymphokine production, but it would be surprising if TCDD did not also suppress their production since it has such a profound effect on other T-cell function. Humoral factors do not appear to be dominant in impaired host resistance to salmonella infection in mice since full protection from challenge appears to depend upon the immune response that leads to delayed-type hypersensitivity, rather than to that which results in circulating antibody and arthus sensitivity (4). The effect of TCDD on humoral antibody production in mice is not known. Recent studies by one of the authors (R. E. Faith, unpublished data) indicates that TCDD has no effect on humoral antibody production to bovine gamma globulin in rats. In contrast, the guinea pig humoral response to tetanus toxoid was observed to be impaired (Vos et al. [29]).
VOL. 12, 1975
TCCD-DECREASED HOST RESISTANCE TO INFECTION
Alterations in the functions of phagocytic cells, either polymorphonuclear or mononuclear, may be involved. This could result from necrosis of these cells and/or a decrease in production or blockage of functional abilities of these cells. A recent study by Weissberg and Zinkl (33) indicated no significant change in peripheral blood monocyte numbers in rats treated with TCDD. In addition, these authors report an elevation in peripheral blood neutrophil counts after TCDD treatment. This would suggest that the defect in host resistance is not the result of destruction of circulating phagocytes. However, this does not rule out the possibility of death or destruction of tissue-fixed macrophages. The number of circulating monocytes is small in relation to those that are tissue fixed, and the study of circulating monocytes does not necessarily reflect the state of production or function of mononuclear phagocytes. In this light, it is of interest to note the results of two studies (7, 24) which show that treatment with agents that interfere with monocyte production also suppress host resistance to bacterial infection. Studies designed to investigate the possibilities of alteration in functional abilities of phagocytic cells and production of monocytes in relation to dioxin exposure are being undertaken in this laboratory. In addition, possible defect(s) in cooperation between T cells and phagocytic cells are being investigated. The results of these studies should lend insight into the mechanism(s) of suppressed host resistance to infection induced by exposure to TCDD. ACKNOWLEDGMENTS We wish to thank Mary Clements, Martha W. Harris, and Janet D. Allen for excellent technical assistance and J. K. Haseman for statistical analyses. LITERATURE CITED 1. Bakken, K., and T. M. Vogelsang. 1950. The pathogenesis of Salmonella typhimurium infection in mice. Acta Pathol. Microbiol. Scand. 27:41-50. 2. Bartholomew, D. J. 1959. A test of homogeneity for ordered alternatives. Biometrika 46:36-48. 3. Carter, C. D., R. D. Kimbrough, J. A. Liddle, R. E. Cline, M. M. Zack, Jr., W. F. Barthel, R. E. Koehler, and P. E. Phillips. 1975. Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188:738-740. 4. Collins, F. M., and G. B. Mackaness. 1968. Delayed
hypersensitivity and arthus reactivity in relation to host resistance in Salmonella-infected mice. J. Immunol. 101:830-845. 5. Firestone, D. 1973. Etiology of chick edema disease. Environ. Health Perspect. 5:59. 6. Friend, M., and D. 0. Trainer. 1970. Polychlorinated biphenyl: interaction with duck hepatitis virus. Science 170:1314-1316. 7. Gadeberg, 0. V., J. M. Rhodes, and S. 0. Larsen. 1975.
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The effect of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli 04:K3:H5 and degradation of "'I-labeled HSA-antibody complexes by these cells. Immunology 28:59-70. 8. Gainer, J. H. 1972. Increased mortality in encephalomyocarditis virus-infected mice consuming cobalt sulfate: tissue concentrations of cobalt. Am. J. Vet. Res. 33:2067-2073. 9. Gainer, J. H., J. Long, Jr., P. R. Hill, and W. I. Capps. 1971. Inactivation of the pseudorabies virus by dithiothreitol. Virology 45:91-100. 10. Gainer, J. H.,and T. W. Pry. 1972. Effects of arsenicals on viral infections in mice. Am. J. Vet. Res.33:22992307. 11. Hemphill, F. E., M. L. Kaeberle, and W. B. Buck. 1971. Lead suppression of mouse resistance to Salmonella typhimurium. Science 172:1031-1032. 12. Hollander, M., and D. A. Wolfe. 1973. Nonparametric statistical methods. John Wiley and Sons, New York. 13. Jonckheere, A. R. 1954. A distribution-free k-sample test against ordered alternatives. Biometrika 41:133. 14. Jones, R. H., R. L. Williams, and A. M. Jones. 1971. Effects of heavy metal on the immune response. Preliminary findings for cadmium in rats. Proc. Soc. Exp. Biol. Med. 137:1231-1236. 15. Kantor, F. S. 1975. Infection, anergy and cell-mediated immunity. N. Engl. J. Med. 292:629-634. 16. Kimmig, J., and K. H. Schulz. 1957. Berufliche Akne (sog. chlorakne) durch chlorierte aromatische zyklische ather. Dermatologica 115:540. 17. Koller, L. D. 1973. Immunosuppression produced by lead, cadmium and mercury. Am. J. Vet. Res. 34:1457-1458. 18. Koller, L. D., and S. Kovacic. 1974. Decreased antibody formation in mice exposed to lead. Nature (London) 250:148-150. 19. Koller, L. D., and J. E. Thigpen. 1973. Reduction of antibody to pseudorabies virus in polychlorinated biphenyl-exposed rabbits. Am. J. Vet. Res. 34:16051606. 20. Lane, F. C., and E. R. Unanue. 1972. Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135:1104-1112. 21. McFerran, J. B., and C. Dow. 1970. Experimental Aujeszky's disease (pseudorabies) in rats. Br. Vet. J. 126:173-179. 22. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 23. Mauel, J. 1974. Cell-mediated immune mechanisms in bacterial and protozoal infections. Prog. Immunol. II 4:109-116. 24. North, R. J. 1970. Suppression of cell-mediated immunity to infection by an antimitotic drug. J. Exp. Med. 132:535-545. 25. Ruskin, J., J. McIntosh and J. S. Remington. 1969. Studies on the mechanisms of resistance to phylogenetically diverse intracellular organisms. J. Immunol. 103:252-259. 26. Smith, A. W. 1973. The mouse. Aeromed. Rev. 19:1115. 27. Verschuuren, H. G., E. J. Ruitenberg, F. Peetoom, P. W. Helleman, and G. J. van Esch. 1970. Influence of triphenyltin acetate on lymphatic tissue and immune responses in guinea pigs. Toxicol. Appl. Pharmacol. 16:400-410. 28. Vos, J. G., and J. A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Arch. Allergy 47:777-794. 29. Vos, J. G., J. A. Moore, and J. G. Zinkl. 1973. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune
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system of laboratory animals. Environ. Health Perspect 5:149-162. 30. Vos, J. G., and H. van Genderen. 1973. Toxological aspects of immunosuppression, p. 527-545. In Pesticides and the environment: a continuing controversy. Symposia Specialists, Miami. 31. Vos, J. G., J. A. Moore, and J. G. Zinkl. 1974. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29:229-241.
INFECT. IMMUN. 32. Wassermann, M., D. Wassermann, Z. Gershon, and L. Zellermayer. 1969. Effects of organochlorine insecticides on body defense systems. Ann. N.Y. Acad. Sci. 160:393-401. 33. Weissberg, J. S., and J. G. Zinkl. 1973. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin upon hemostasis and hematalogic function in the rat. Environ. Health Perspect 5:119-123.
INFECTION AND IMMUNITY, Dec. 1975, p. 1319-1324 Copyright©) 1975 American Society for Microbiology
Increased Susceptibility to Bacterial Infection as a Sequela of Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J. E. THIGPEN,* R. E. FAITH, E. E. McCONNELL, AND J. A. MOORE National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received for publication 12 August 1975
The effects of subclinical levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) the response of mice to infection with either Salmonella bern or Herpesvirus suis, also known as pseudorabies virus, are reported. TCDD is a contaminant of certain commercially useful chemicals, such as chlorinated phenols or herbicides. It has been shown to cause thymic atrophy and to suppress cell-mediated immunity in laboratory animals. Sublethal levels of TCDD were used: 0.5, 1, 5, 10, or 20 Mg/kg, given through a gastric tube once weekly for 4 weeks. A significant decrease in weight gain compared with control mice occurred at the 20-,ug dosage. Dose schedules of 1 ,ug or more, followed by salmonella infection, resulted in significant increases in mortality and decreases in the time from infection to death. However, TCDD had no significant effect on mortality in the pseudorabies-infected mice. The most important finding in this study is that extremely low levels of TCDD, which do not produce clinical or pathological change, still have the capacity to affect host defense. on
Various environmental chemicals have been shown to affect immune responses or host resistance to infectious agents (Friend and Trainer [6], Gainer [81, Gainer and Pry [10], Hemphill et al. [11], Jones et al. [14], Koller and Thigpen [191, Koller and Kovacic [18], Koller [171, Verschuuren et al. [27], Vos and van Genderen [30], Vos et al. [291, Vos and Moore [28], Wassermann et al. [32]). These reports generally involve observed defects in antibody response (14, 17, 18, 19, 27, 30, 32) or on cell mediated immunity (28, 29, 30), although several (6, 8, 10, 11) also describe suppressed host resistance to infection. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) can occur as a highly toxic contaminant in the production of some chlorinated phenols or chemicals synthesized from chlorinated phenols such as the herbicide 2,4,5-trichlorophenoxyacetic acid and thus could be unknownly distributed in the environment. TCDD as an environmental contaminant can present health problems, both in man and other animals. It is associated with chloracne in man (16) and chick edema disease in chickens (5). More recently TCDD and 2,4,5-trichlorophenol were implicated as the probable cause of toxicity in a group of horses in eastern Missouri. In addition to the horses, insects, birds, and rodents were poisoned, and there were several cases of human illness attributed to TCDD in this incident (3). TCDD has been observed to cause profound thymic atrophy and suppress cell-mediated im-
munity in rats, mice, and guina pigs (28, 29). It is not known whether TCDD-exposed animals are more susceptible to infectious agents. These studies were designed to determine the effects of subclinical levels of TCDD on the response of mice subsequently infected with either Salmonella bern or pseudorabies virus (PRV). The criteria used to measure these effects were clinical appearance, weight gains, mortality rates, time from infection to death, and pathological
examination. MATERIALS AND METHODS Animal. Four-week-old male C57BL/6Jfh (J67) specific-pathogen-free mice were purchased from
Jackson Laboratories, Bar Harbor, Me. The mice were caged individually under filter tops (Envirogard, Lab Products, Garfield, N.J.) following a rigid sanitary regimen and given food (Sterilizable LabBlox, Allied Mills, Inc., Chicago, Ill.) and water ad libitum. TCDD. TCDD, greater than 99% purity (lot 851144-II), was a gift from Dow Chemical Company, Midland, Mich. It was dissolved in reagent-grade acetone and subsequently diluted with at least 6 parts of corn oil. The exact volume of corn oil was adjusted to provide each mouse with a dose volume of approximately 0.2 ml. Infectious agents. The bacterium used was a S. bern isolate from a naturally infected opossum. This organism was grown overnight at 35 to 37 C on Trypticase soy agar containing 5% sheep blood. Bacterial dilutions were prepared in sterile physiological saline, and the number of bacteria was quanti-
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INFECT. IMMUN.
THIGPEN ET AL.
tated turbidimetrically with a Coleman model 6/20 spectrophotometer at 540 ,um. In addition, the number of total viable bacteria in all dilutions used for animal inoculation was confirmed by conventional total plate count procedures. The viral agent used was Herpesvirus suis (PRV) (9). PRV was produced in 75-cm2 Falcon flasks containing RK-13 monolayers. After a 4-plus cytopathogenic effect was observed, the medium from each flask was centrifuged at 800 x g to remove large cellular debris. The supernatants from each flask were pooled, thoroughly mixed, aliquoted in 2-ml vials, and stored at -79 C until used. At the time of inoculation the original suspension contained approximately 5.0 x 10' tissue culture infective doses/ ml. The pooled viral suspension was culture negative for mycoplasma and bacteria. Preliminary study. Lethal dose response curves were established for S. bern and PRV to determine challenge doses (0.2 ml intraperitoneally [i.p.]) that would produce 20% mortality in 8-week-old normal mice. All mice were observed for 14 days. Dead mice were examined by both microbiological and histopathological procedures to verify infection. Experimental design. Two different experiments were performed. In each experiment 4-week-old mice were randomly divided into four groups. Each mouse was weighed and dosed by gavage once each week for 4 consecutive weeks. Mice were dosed with either TCDD in acetone-corn oil or with acetone-
nation were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 ,um, and routinely stained with hematoxylin and eosin. The criteria used to compare the effects of sublethal levels of TCDD in mice subsequently infected with S. bern or PRV were clinical appearance, weight gains, mortality rates, time from infection to death, and pathological examination. Statistical analyses. For the mortality data, dose response trends were analyzed by the Bartholomew test (2), and pair-wise comparisons were made by the Fisher exact test for 2 x 2 tables (12). For the other variables, the significance of dose-response trends was determined by the Jonckheere test (13), and treatment-control differences were evaluated by the two-sided Mann-Whitney ,u tests (12).
RESULTS Effects of TCDD on weight gain, mortality rate, and time from infection to death. There was essentially no difference in mean group body weights prior to TCDD dosing. By week 4, the high-dose group (20 gg of TCDD per kg) gained significantly (P < 0.01) less weight than did the controls. The 10 jig of TCDD per kg dose group also gained less weight; all other TCDD group body weights were comparable to the group which received acetone-corn oil (Table 1). At a given level of TCDD there were no significorn oil. cant differences in the week 4 weights among In the first experiment, each mouse received the mice assigned to the bacteria, virus, or either 0, 5, 10, or 20 ,ug of TCDD/kg per week. Two control groups. There was little association bedays after the last dose of TCDD, when the mice tween body weight at the time of infection and were 8 weeks old, each group was randomly divided into three subgroups. Mice in one subgroup were subsequent survival. Mortality rates in the bacteria and virus coninjected i.p. with 10' viable S. bern organisms (LD20114 dose), the second subgroup received 104 TCID trol groups that had not received TCDD were as of PRV (LD2014 dose), and the third subgroup (con- anticipated based on the LD2014 preliminary trol) was injected with saline only. (LD204,, is the study (Table 2). The groups of mice exposed to number of bacterial or viral agents required to kill 1, 5, 10, or 20 ,ug of TCDD per kg and given S. approximately 20% of the mice inoculated within a bern showed a significant (P < 0.05 to 0.01) 14-day observation period.) The second experiment was conducted in a man- increase in mortality rates. The mortality rate identical to the first except that each mouse received either 0, 0.5, 1, or 5 jig of TCDD/kg per week and the PRV subgroup was eliminated. Both experiments were performed as blind studies, and the order of inoculation was randomized. Neither the bacterium nor the virus appeared to lose or gain potency during the time of administration. The level of mortality seen in the final 25% of the animals inoculated was not statistically different from that observed in the first 25% or the middle 50%. Therefore, the order of inoculation was virtually irrelevant to subsequent survival. After bacterial and viral challenge, the animals were observed at least twice daily for 14 days. Body weights were recorded at death or at termination of the study. Necropsies were performed on at least three mice from each subgroup, and selected tissues were obtained for histopathological and microbiological examination. Mice from subgroups with less than three deaths were sacrificed and necropsied on day 14. The tissues selected for histopathological exami-
ner
TABLE 1. Effect of TCDD on weight gain of mice over a 3-week exposure period Weekly TCDD dosef (g/kg of body wt)
No.
No.
Initial wt (g)b
Wt gain hleg at challenge a
ofe micl()
Expt I 20 10 5 0
Expt II 5 1 0.5 0
60 59 60 60
18.68 18.78 18.41 18.65
± 0.21 ± 0.22
35 33 35 35
20.81 21.08 20.67 21.13
+ + + +
+ 0.22 + 0.19 0.21 0.22 0.24 0.24
3.06 0.20 5.21 + 0.17 5.77 + 0.19 5.65 + 0.17
4.56 4.50 4.06 4.29
+ + + +
0.17 0.17 0.19 0.14
' TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. bMean ± standard error. Cp < 0.01.
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TCCD-DECREASED HOST RESISTANCE TO INFECTION
VOL. 8, 1975
in the 0.5 ug of TCDD per kg dose group was identical to the control group; therefore, the "no effect" level based on the parameters of this study would be somewhere between 0.5 and 1.0 ,ug of TCDD per kg (Table 2; Fig. 1). The mortality rates in the groups that re-
ceived virus were not seen to differ significantly with variations in the dose of TCDD. There was a significant (P < 0.01) dose-related decrease in time from infection to death in the S. bern-challenged mice (Table 3; Fig. 1).
TABLE 2. Comparison of 14-day mortality rates of mice exposed to sublethal levels of TCDD and challenged with either S. bern or PRV Weekly Mortality rates (%) TCDD dosea
(/Ag/kg
of body wt) Expt I 20 10 5 0
Viral
Bacterial challengeb
challengee
95 (19/20)'-f 65 (13/20)° 65 (13/20)9 25 (5/20)
10 (2/20) 0 (0/20) 15 (3/20) 15 (3/20)
Experiment I 80 _ _ __ _
60k
Controld 0 0 0 0
(0/20) (0/19) (0/20) (0/20)
Expt II ND, 0 (0/15) 70 (14/20)9 5 8 (1/13) ND 1 65 (13/20)9 0 (0/15) ND 25 (5/20) 0.5 ND 0 (0/15) 0 25 (5/20) a TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. bBacterial challenge (LD20114) of S. bern via i.p. injection on day 2 after the 4th weekly dose of TCDD. c Viral challenge (LD20114) of PRV via i.p. injection on day 2 after the 4th weekly dose of TCDD. d Control animals were injected i.p. with sterile saline on day 2 after the 4th weekly dose of TCDD. ' The numbers in parentheses indicate the number of mice dying per the total number of mice in the group. fP < 0.01. oP < 0.05. hND, Not done.
*20 ,pg/kg AIO pg/kg @5 pg/kg 0O ug/kg
40
2
20
E
A_ u
>
_
_
-
f1
Experiment I
E
80K
60K
E
1
0 z40-
A 50 .pg/kg ... .I A 1.0 pg/kg 0* 0.5 pg/kg
>
-
-
fK
o000 Ag/kg
/
220 _
0
/ 2
6 8 Time (days)
4
12
10
14
FIG. 1. Cumulative mortality rates and time from infection to death in mice exposed to sublethal levels of TCDD and subsequently infected with S. bern.
TABLE 3. Average time from infection to death (days) Viral challenge'
Bacterial challengeb
Weekly TCDD dosea (jIg/kg of body wt)
No. of
nonsurvivorsd Expt I 20 10 5 0
Time to deathe
19 13 13 5
2.95 0.49f 5.69 0.67 4.54 + 0.849 8.40 1.69
14 13 5 5
2.86 6.08 9.00 7.40
No. of
nonsurvivorsd 2 0 3 3
Time to death"
9.00 + 5.00
6.33 4.67
1.21 0.33
Expt II 5.0 1.0 0.5 0
0.38' 0.92 1.95 1.81
NDh ND ND ND
TCDD in acetone-corn oil carrier via gastric intubation on an individual body weight basis. Bacterial challenge (LD20114) of S. bern via i.p. injection on day 2 after the 4th weekly dose of TCDD. c Viral challenge (LD20,,4) of PRV via i.p. injection on day 2 after the 4th weekly dose of TCDD. d The number of nonsurvivors was the number of animals dying within 14 days after bacterial or viral challenge. e X standard error. a
b
fP < 0.01. 9p < 0.05. h
ND, Not done.
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THIGPEN ET AL.
Pathological observations. Postmortem examination of mice treated with TCDD only revealed dose-dependent lesions in the liver and thymus similar to those described by Vos et al. (31). The thymus from mice in the 20 ,ug/kg group was smaller than normal. Microscopic examination revealed cortical atrophy due to loss of small lymphocytes. Thymic atrophy was much less apparent and often equivocal in the 10 pLg/kg group and was not observed at the lower doses. Interestingly, no lesions were found in the spleen. The livers from the 20 ,tg/kg group were lighter in color than normal and were mottled. This was less evident in the 10 ,ug/kg animals, and no macroscopic lesions were visible in the livers from mice in the 5, 1, or 0.5 ,ug of TCDD per kg dose groups. Histopathological examination of livers from two high-dose groups revealed a nodular surface with variable degrees of hepatocellular cytomegaly and occasional foci of single cell necrosis. A few inflammatory cells (predominantly mononuclear) were found in the areas of necrosis. In addition, there were often intracellular accumulations oflipid in hepatocytes, especially in midzonal areas. Lesions were not observed at lower doses or in other organs.
The lesions described were still apparent in the mice from the two highest dose groups inoculated with virus or bacteria (both survivors and nonsurvivors). In addition, mice in the bacterial groups that died showed macro- and microscopic lesions typical of salmonellosis (1, 26). There was widespread infiltration of polymorphonuclear leukocytes, particularly in visceral organs. Focal necrotic hepatitis resembling microinfarction was attributed to adjacent areas of vasculitis and thrombosis. Lymphoid necrosis, thrombotic vasculitis, and suppuration were also observed in the spleen. Necrotic enteritis, purulent peritonitis, and occasionally suppurative meningitis were also noted. In the virus portion of the experiment, lesions attributable to PRV were not observed in any of the mice examined. PRV in laboratory rodents is known to produce few lesions at the light microscopic level (21).
DISCUSSION The data presented in this study clearly established that mice exposed to four weekly doses of 1, 5, 10, or 20 ,ug of TCDD per kg and subsequently infected with an LD20/14 dose of S. bern have a significantly higher rate of mortality with a shortened incubation period. Furthermore, the mice exposed to 1 or 5 ,ug of TCDD per kg appeared clinically normal, had a normal
INFECT. IMMUN.
rate of weight gain, and showed no histopathological evidence of toxicity. This demonstrates the most important aspect of this study, i.e., that extremely low levels of TCDD which do not produce clinical or pathological change still have the capacity to affect host defense. These findings are significant in that humans may be exposed to environmental contaminants such as TCDD in minute quantities and therefore may be rendered more susceptible to certain types of infectious agents. The fact that TCDD did not increase mortality in mice challenged with PRV suggests a difference in the pathogenesis of these two agents. The reason(s) for this difference is not understood. There are several host defense factors which could account for the increased susceptibility to a facultative intracellular organism such as S. bern. These include: (i) a defect in cell-mediated immunity either through loss of mediating cells or by inhibition of cell function; (ii) a defect on the humoral system; (iii) a defect in one or more of the steps of phagocytosis; and (iv) a reduction or loss of phagocytic cells. Cell-mediated immune reactions as evidenced by delayed hypersensitivity have been shown to play an important role in immunity and recovery from infections with salmonella and other facultative intracellular organisms (4, 15, 20, 22, 23, 25). Delayed hypersensitivity is thought to be mediated through production of lymphokines by specifically activated T cells in response to bacterial antigens (4, 20, 22, 23, 25). TCDD has been shown to suppress delayed hypersensitivity reactions and responses of T cells to activation by the mitogens concanavalin A and phytohemagglutinin (28, 29). These studies did not investigate the influence of TCDD on lymphokine production, but it would be surprising if TCDD did not also suppress their production since it has such a profound effect on other T-cell function. Humoral factors do not appear to be dominant in impaired host resistance to salmonella infection in mice since full protection from challenge appears to depend upon the immune response that leads to delayed-type hypersensitivity, rather than to that which results in circulating antibody and arthus sensitivity (4). The effect of TCDD on humoral antibody production in mice is not known. Recent studies by one of the authors (R. E. Faith, unpublished data) indicates that TCDD has no effect on humoral antibody production to bovine gamma globulin in rats. In contrast, the guinea pig humoral response to tetanus toxoid was observed to be impaired (Vos et al. [29]).
VOL. 12, 1975
TCCD-DECREASED HOST RESISTANCE TO INFECTION
Alterations in the functions of phagocytic cells, either polymorphonuclear or mononuclear, may be involved. This could result from necrosis of these cells and/or a decrease in production or blockage of functional abilities of these cells. A recent study by Weissberg and Zinkl (33) indicated no significant change in peripheral blood monocyte numbers in rats treated with TCDD. In addition, these authors report an elevation in peripheral blood neutrophil counts after TCDD treatment. This would suggest that the defect in host resistance is not the result of destruction of circulating phagocytes. However, this does not rule out the possibility of death or destruction of tissue-fixed macrophages. The number of circulating monocytes is small in relation to those that are tissue fixed, and the study of circulating monocytes does not necessarily reflect the state of production or function of mononuclear phagocytes. In this light, it is of interest to note the results of two studies (7, 24) which show that treatment with agents that interfere with monocyte production also suppress host resistance to bacterial infection. Studies designed to investigate the possibilities of alteration in functional abilities of phagocytic cells and production of monocytes in relation to dioxin exposure are being undertaken in this laboratory. In addition, possible defect(s) in cooperation between T cells and phagocytic cells are being investigated. The results of these studies should lend insight into the mechanism(s) of suppressed host resistance to infection induced by exposure to TCDD. ACKNOWLEDGMENTS We wish to thank Mary Clements, Martha W. Harris, and Janet D. Allen for excellent technical assistance and J. K. Haseman for statistical analyses. LITERATURE CITED 1. Bakken, K., and T. M. Vogelsang. 1950. The pathogenesis of Salmonella typhimurium infection in mice. Acta Pathol. Microbiol. Scand. 27:41-50. 2. Bartholomew, D. J. 1959. A test of homogeneity for ordered alternatives. Biometrika 46:36-48. 3. Carter, C. D., R. D. Kimbrough, J. A. Liddle, R. E. Cline, M. M. Zack, Jr., W. F. Barthel, R. E. Koehler, and P. E. Phillips. 1975. Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188:738-740. 4. Collins, F. M., and G. B. Mackaness. 1968. Delayed
hypersensitivity and arthus reactivity in relation to host resistance in Salmonella-infected mice. J. Immunol. 101:830-845. 5. Firestone, D. 1973. Etiology of chick edema disease. Environ. Health Perspect. 5:59. 6. Friend, M., and D. 0. Trainer. 1970. Polychlorinated biphenyl: interaction with duck hepatitis virus. Science 170:1314-1316. 7. Gadeberg, 0. V., J. M. Rhodes, and S. 0. Larsen. 1975.
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The effect of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli 04:K3:H5 and degradation of "'I-labeled HSA-antibody complexes by these cells. Immunology 28:59-70. 8. Gainer, J. H. 1972. Increased mortality in encephalomyocarditis virus-infected mice consuming cobalt sulfate: tissue concentrations of cobalt. Am. J. Vet. Res. 33:2067-2073. 9. Gainer, J. H., J. Long, Jr., P. R. Hill, and W. I. Capps. 1971. Inactivation of the pseudorabies virus by dithiothreitol. Virology 45:91-100. 10. Gainer, J. H.,and T. W. Pry. 1972. Effects of arsenicals on viral infections in mice. Am. J. Vet. Res.33:22992307. 11. Hemphill, F. E., M. L. Kaeberle, and W. B. Buck. 1971. Lead suppression of mouse resistance to Salmonella typhimurium. Science 172:1031-1032. 12. Hollander, M., and D. A. Wolfe. 1973. Nonparametric statistical methods. John Wiley and Sons, New York. 13. Jonckheere, A. R. 1954. A distribution-free k-sample test against ordered alternatives. Biometrika 41:133. 14. Jones, R. H., R. L. Williams, and A. M. Jones. 1971. Effects of heavy metal on the immune response. Preliminary findings for cadmium in rats. Proc. Soc. Exp. Biol. Med. 137:1231-1236. 15. Kantor, F. S. 1975. Infection, anergy and cell-mediated immunity. N. Engl. J. Med. 292:629-634. 16. Kimmig, J., and K. H. Schulz. 1957. Berufliche Akne (sog. chlorakne) durch chlorierte aromatische zyklische ather. Dermatologica 115:540. 17. Koller, L. D. 1973. Immunosuppression produced by lead, cadmium and mercury. Am. J. Vet. Res. 34:1457-1458. 18. Koller, L. D., and S. Kovacic. 1974. Decreased antibody formation in mice exposed to lead. Nature (London) 250:148-150. 19. Koller, L. D., and J. E. Thigpen. 1973. Reduction of antibody to pseudorabies virus in polychlorinated biphenyl-exposed rabbits. Am. J. Vet. Res. 34:16051606. 20. Lane, F. C., and E. R. Unanue. 1972. Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135:1104-1112. 21. McFerran, J. B., and C. Dow. 1970. Experimental Aujeszky's disease (pseudorabies) in rats. Br. Vet. J. 126:173-179. 22. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 23. Mauel, J. 1974. Cell-mediated immune mechanisms in bacterial and protozoal infections. Prog. Immunol. II 4:109-116. 24. North, R. J. 1970. Suppression of cell-mediated immunity to infection by an antimitotic drug. J. Exp. Med. 132:535-545. 25. Ruskin, J., J. McIntosh and J. S. Remington. 1969. Studies on the mechanisms of resistance to phylogenetically diverse intracellular organisms. J. Immunol. 103:252-259. 26. Smith, A. W. 1973. The mouse. Aeromed. Rev. 19:1115. 27. Verschuuren, H. G., E. J. Ruitenberg, F. Peetoom, P. W. Helleman, and G. J. van Esch. 1970. Influence of triphenyltin acetate on lymphatic tissue and immune responses in guinea pigs. Toxicol. Appl. Pharmacol. 16:400-410. 28. Vos, J. G., and J. A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. Arch. Allergy 47:777-794. 29. Vos, J. G., J. A. Moore, and J. G. Zinkl. 1973. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune
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system of laboratory animals. Environ. Health Perspect 5:149-162. 30. Vos, J. G., and H. van Genderen. 1973. Toxological aspects of immunosuppression, p. 527-545. In Pesticides and the environment: a continuing controversy. Symposia Specialists, Miami. 31. Vos, J. G., J. A. Moore, and J. G. Zinkl. 1974. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29:229-241.
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