(Treponema) hyodysenteriae Pathogenesis - Infection and Immunity

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Dec 23, 1991 - Bacteroides vulgatus, and Fusobacterium necrophorum. J. Am. Vet. Med. Assoc. 172:468-471. 6. Harris, D. L., R. D. Glock, C. R. Christensen, ...
Vol. 60, No. 8

INFECrION AND IMMUNITY, Aug. 1992, p. 3433-3436

0019-9567/92/083433-04$02.00/0 Copyright X) 1992, American Society for Microbiology

An Enhanced Murine Model for Studies of Serpulina (Treponema) hyodysenteriae Pathogenesis S. K. NIBBELINK AND M. J. WANNEMUEHLER* Department of Microbiology, Immudology, and Preventive Medicine, Veterinary Medical Research Institute,

Iowa State University, Ames, Iowa 50011 Received 23 December 1991/Accepted 3 April 1992

A defined diet was used to increase the susceptibility of mice to Serpulina hyodysenteriae. BALB/cByJ, C3H/HeN, and C3H/HeJ mice, when fed the defined diet 7 to 14 days prior to and throughout the challenge period, consistently showed higher incidences of disease than mice maintained on normal rodent chow. The use of this defined diet will increase the consistency of in vivo studies following infection with S. hyodysenteriae in the mouse model. Swine dysentery is a mucohemorrhagic diarrheal disease of growing swine; it affects the cecum and colon (1, 6, 8). Although the etiologic agent, Serpulina (Treponema) hyodysentenae, has been described (6), the mechanism(s) of pathogenesis is unknown. Potential virulence determinants of S. hyodysentenae are a lipopolysaccharide or lipopolysaccharidelike moiety (2-4, 19, 20) and a ,-hemolysin (14, 21). A mouse model of S. hyodysentenae infection exists; however, care should be exercised in extrapolating phenomena observed in the mouse to those in the natural host, the pig. The murine model, which was initially described by Joens and Glock (10), is characterized by catarrhal typhlitis. Murine cecal histopathological changes are similar to pathological changes observed in swine (10); however, clinical signs in the mouse are diminished when compared with those in swine. The mouse model has been used extensively in pathogenesis research of S. hyodysentenae (7, 10-12, 16-18, 20, 22) but the inconsistency of disease in the mouse model often makes interpretation of results difficult, especially for certain strains of mice (18). Differences in disease susceptibility have been described as a result of genetic differences (18, 20) and the source of the mice (22). In this paper we describe the use of a defined diet which increases the susceptibility of mice to infection by S. hyodysenteriae. BALB/cByJ, C3H/HeSnJ, and C3H/HeJ mice (originally procured from Jackson Laboratory, Bar Harbor, Maine) and C3HIHeN mice (originally obtained from Harlan Sprague Dawley, Indianapolis, Ind.) were obtained from breeding colonies maintained at Iowa State University. The inbred mouse strains used in this study were selected to compare the effect of the diet on a resistant and a susceptible mouse strain; it was previously shown (18) that BALB/cByJ mice and C3H strains were refractory and susceptible, respectively, to development of cecitis after S. hyodysenteriae challenge. All breeding colonies and principals of these trials were maintained on Mouse Lab Chow 5010 (nutritionally complete) (Purina Mills, Inc., St. Louis, Mo.) until experimentation. For the sake of convenience, Mouse Lab Chow will be referred to herein as "normal diet." Mice were 3 to 5 months old at time of experimentation. S. hyodysenteriae B204 was cultivated in broth as previously described (18). Some mice were fed Teklad Diet TD 85420 (Harlan Sprague Dawley, Madison, Wis.), a nutritionally complete defined *

Corresponding author. 3433

diet, for 7 to 14 days prior to and throughout the challenge period. Mice were orally challenged by gastric intubation on two consecutive days, concurrent with a 36-h fast, with various doses of S. hyodysentenae B204 as previously described (18). The mice were necropsied at 10 to 20 days postchallenge, and the ceca were visualized for development of grossly evident lesions. Cecal lesions were scored as previously described (18); briefly, a cecum with no grossly evident lesions was scored 0; one with excess intraluminal mucus was scored 1; one exhibiting excess intraluminal mucus and organ atrophy in the cecal apex only was scored 2; and one with excess cecal intraluminal mucus and organ atrophy was scored 3. To determine colonization, cecal swabs were cultured on blood agar plates containing antibiotics (13, 18). In some trials, the number of CFU per gram of cecal weight was enumerated by serial dilution and pour plating (18). Experiment 1 (Table 1) illustrates the susceptibility of C3HJHeN mice to S. hyodysenteriae when maintained on Teklad diet TD 85420. Mice fed the Teklad diet showed increased mean cecal scores (greater severity of cecal lesions) (scores of 3.0, 2.6, and 3.0 for challenge doses of 106, 105, and 104 organisms, respectively) compared with previously published data of similarly challenged C3H/HeN mice fed a normal diet (scores of 2.1, 1.8, and 1.3 for challenge doses of 106, 105, and 104 organisms, respectively) (18). Experiment 2 (Table 1) shows that at challenge doses of 108, 107, and 106 spirochetes, C3HIHeJ mice developed more consistent lesions (3.0, 3.0, and 2.6 versus 2.1, 1.2, and 1.4) and higher levels of CFU per gram of cecum (8.72 to 8.89 versus 8.00 to 6.51) than previously described for C3H/HeJ mice maintained on normal rodent chow (18). Furthermore, C3H/HeJ mice maintained on the Teklad diet developed cecal lesions after infection with as few as 100 organisms (experiment 3, Table 1). This is in contrast to previous results (18), which indicated that for C3H/HeJ mice, the infectious dose which yielded a lesion score of 3 for 50% of challenged mice was 8.3 x 107 S. hyodysenteriae B204 organisms for mice maintained on the normal diet. BALB/cByJ mice have been described as being refractory to challenge with S. hyodysentenae (18). Figures 1 and 2 illustrate that BALB/cByJ mice, fed the Teklad diet, develop lesions after challenge with S. hyodysenteriae. BALB/cByJ mice maintained on the normal mouse chow diet exhibited no gross lesions at challenge doses of 108 or 107 spirochetes

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NOTES

INFECT. IMMUN.

TABLE 1. Susceptibilities of mice maintained on Teklad diet TD 85420 to challenge with S. hyodysenteriae B204

Expt

ProtoCola

Bacterial dose (organisms)6

mean cecal scorec Group(no. in group)

Logl0 CFU/gd

7 days, 20 dpi

106

105 104

3.0 ± 0 (5) 2.6 ± 0.4 (5) 3.0 0 (5)

7.95 8.29 8.08

108 107

3.0 ± 0 (4) 3.0 ± 0 (5) 2.6 ± 0.4 (5)

8.72 8.88 8.89

± 0 (7) ± 0.5 (5) ± 0.7 (5)

NDe ND ND ND

Mouse strain

C3H/HeN

1

C3H/HeJ

2

14 days, 10 dpi

106 3

C3H/HeJ

14 days, 10 dpi

105 104 103

3.0 1.8 1.8 2.7

102

+ 0.3 (6)

a Mice were maintained on Teklad diet TD 85420 7 or 14 days prior to and throughout the challenge period with S. hyodysenteniae B204. In each experiment, all mice were sacrificed at either 10 or 20 days postinfection (dpi). b Mice were challenged per os with the indicated dose of log-phase S. hyodysenteriae B204 once a day on two consecutive days. Group mean cecal scores are based on grossly observable lesions ranging from 0 (no lesions present) to 3 (severe cecal lesions) ± standard error. d S. hyodysenteriae was reisolated from cecal contents of all mice in each group. Estimation of S. hyodysenteriae colonization density was obtained by enumerating S. hyodysenteriae CFU per gram of cecum. Individual values were determined and are expressed as group mean values. e ND, not determined.

(Fig. 1). Similarly, C3H/HeJ mice, challenged with 106 organisms, developed more severe lesions following S. hyodysentenae challenge when fed the Teklad diet than did those that were fed the normal diet (Fig. 2). Since the BALB/cByJ mice fed the Teklad diet appeared more susceptible to S. hyodysenteniae (Fig. 1 and 2), the infective challenge dose of S. hyodysenteriae for BALB/cByJ mice was determined (Fig. 3). BALB/cByJ mice developed lesions at all challenge doses; however, the susceptibility to challenge was lower than that exhibited by C3H/HeJ mice (Table 1). BALB/cByJ mice (n = 4), maintained on the Teklad TD 85420 diet for 20 days, were injected intraperitoneally with 150 ,g of S. hyodysenteriae whole-cell lysate on day 10. BALB/cByJ mice maintained on the normal diet (n = 4) were also similarly immunized. All mice in both groups were 0%

0%

11-4I n=8

100%

10% 3-

100%

nlprrPnt cuiture r-sltsirp posiuve Tvicitiup percent n=8 11

1

3-

sacrificed 10 days after immunization. Anti-S. hyodysenteniae immunoglobulin responses were not significantly different between the two groups of mice, as determined by enzyme-linked immunosorbent assay (data not shown). The mice maintained on Teklad TD 85420, however, did show an approximate 4-log-unit increase in the number of grampositive organisms recovered from the cecum, as estimated by spread plate counts on phenylethanol agar, and an approximate 2-log-unit increase in the number of cecal gram-negative organisms, as estimated by spread plate counts on eosin-methylene blue (EMB) agar. All culture plates were incubated at 37°C in an aerobic environment

u) cn

C-aed

2-

u

0

CU

5)

u U)

I

u

i 1-

n=10 -

normal

108

4

z

1X~~~~~n1 BALB/cByJ

I

n=13

Percent Culture Positive 100% 82% 100% n=16 n=15

normal

.

Teklad

Teklad

107

108

107

Challenge Dose FIG. 1. Effect of diet on the susceptibility of BALB/cByJ mice to challenge with S. hyodysentenae B204. Mice were maintained on normal feed or Teklad diet TD 85420 for 10 days prior to and throughout the challenge period, challenged per os on two consecutive days with either 108 or 107 bacteria, and sacrificed 15 days postchallenge. Cecal scores represent mean values ranging from 0 (no grossly evident lesions) to 3 (severe organ atrophy with intraluminal mucus). Percent culture-positive values indicate the percentage of individual ceca in each treatment group in which S. hyodysenteriae was reisolated. The standard error of the mean is indicated. n is the group size.

normal

Teklad

Diet

-

normal

C3H/HeJ

Teklad

l10

108

106

160

Infective Challenge

FIG. 2. Effect of diet on the susceptibility of BALB/cByJ and C3H/HeJ mice to challenge with S. hyodysentenae B204. Mice were maintained on normal feed or Teklad diet TD 85420 for 7 days prior to and throughout the challenge period, challenged per os on two consecutive days with either 108 (BALB/cByJ) or 106 (C3H/HeJ) bacteria, and sacrificed 10 days postchallenge. Cecal scores represent mean values ranging from 0 (no grossly evident lesions) to 3 (severe organ atrophy with intraluminal mucus). Percent culturepositive values indicate the percentage of individual ceca in each treatment group in which S. hyodysenteriae was reisolated. The standard error of the mean is indicated. n is the group size.

NOTES

VOL. 60, 1992 Percent Culture Positive 100%

3n=6

~1--

T

100%

83%

100%

67%

n=6

TI

9

3435

hyodysenteriae challenge, causes mice to be more susceptible to lesion development. The use of this diet will result in a more consistent model and will permit the use of genetically diverse strains of inbred mice for the study of the pathogenesis of disease caused by S. hyodysenteriae.

0

C)

This work was supported in part by funds from Zinpro Corp. and U.S. Department of Agriculture grant 88-34116-3762.

1 -n-

REFERENCES

10 103 Challenge Dose 10

102

FIG. 3. Susceptibility of BALB/cByJ mice to decreasing challenge doses of S. hyodysenteriae B204. Mice were maintained on Teklad diet TD 85420 for 7 days prior to and throughout the challenge period, challenged per os on two consecutive days, and sacrificed 10 days postchallenge. Cecal scores represent mean values ranging from 0 (no grossly evident lesions) to 3 (severe organ atrophy with intraluminal mucus). Percent culture-positive values indicate the percentage of individual ceca in each treatment group in which S. hyodysentenae was reisolated. The standard error of the mean is indicated. n is the group size.

1. Argenzio, R. A. 1985. Pathophysiology of swine dysentery, p. 3-19. In C. J. Pfeiffer (ed.), Animal models for intestinal disease. CRC Press, Inc., Boca Raton, Fla. 2. Greer, J. M., and M. J. Wannemuehler. 1989. Comparison of the biological responses induced by lipopolysaccharide and endotoxin of Treponema hyodysenteriae and Treponema innocens. Infect. Immun. 57:717-723. 3. Greer, J. M., and M. J. Wannemuehier. 1989. Pathogenesis of Treponema hyodysenteriae: induction of interleukin-1 and tumor necrosis factor by a treponemal butanol/water extract (endotoxin). Microb. Pathog. 7:279-288. 4. Halter, M. R., and L.A. Joens. 1988. Lipooligosaccharides from Treponema hyodysenteriae and Treponema innocens. Infect. Immun. 56:3152-3156. 5. Harris, D. L., T. J. L. Alexander, S. C. Whipp, I. M. Robinson,

R. D. Glock, and P. J. Matthews. 1978. Swine dysentery: studies

(data not shown). To determine whether a factor(s) in Teklad TD 85420 increased the susceptibility of mice to challenge, C3H/HeSnJ and C3H/HeJ mice, maintained on the normal diet, were fed water containing 0.75 M D-glucose ad libitum 7 days prior to and throughout a challenge period with S. hyodysentenae B204. At 12 days postinfection, no increase in the susceptibility to induction of cecal lesions was observed when compared with mice that had drunk water not supplemented with glucose (data not shown). This would suggest that the inclusion of the major component of the TD 85420 diet (i.e., 63% glucose) in the water of mice maintained on a normal diet is insufficient to enhance susceptibility to disease after infection with S. hyodysenteriae. The reason(s) for the increase in susceptibility to S. hyodysenteriae is unclear, but the Teklad diet appears to facilitate cecal colonization by S. hyodysentenae. A possible explanation for this hypothesis is alteration of the cecal microflora. Cecal microflora composition has been postulated to play a major role in the susceptibility of mice to challenge with S. hyodysenteriae (7, 16, 22). Previous work with gnotobiotic pigs (5, 15, 23) characterized the necessity for other bacteria to be present in conjunction with S. hyodysentenae for lesions to develop. Joens et al. (12) have shown that S. hyodysentenae plus Bacteroides vulgatus induced cecal lesions in gnotobiotic mice; however, S. hyodysenteriae alone or B. vulgatus alone did not induce gross lesions. Hayashi et al. (7) described Bacteroides uniformis as a cecal flora component which facilitates infection of Ta:CF-1 mice by S. hyodysentenae. Additionally, Suenaga and Yamazaki (22) reported that mice obtained from different suppliers had different susceptibilities to S. hyodysenteniae and theorized that differing gut floras of mice derived from different breeding colonies (9) were affecting lesion development. Kennedy et al. (12a) and Nibbelink (16) have described a mouse model of S. hyodysenteniae infection involving oral pretreatment of mice with antibiotics. In toto, these results indicate the importance of the character of the resident intestinal microflora in the development of lesions after challenge with S. hyodysenteriae. The Teklad diet TD 85420, when fed to mice before S.

of gnotobiotic pigs inoculated with Treponema hyodysenteriae,

6.

7.

8. 9. 10. 11. 12.

Bacteroides vulgatus, and Fusobacterium necrophorum. J. Am. Vet. Med. Assoc. 172:468-471. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon. 1972. Swine dysentery-1. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin. 67:61-68. Hayashi, T., I. Suenaga, T. Komeda, and T. Yamazaki. 1990. Role of Bacteroides uniformis in susceptibility of Ta:CF*1 mice to infection by Treponema hyodysenteriae. Zentralbl. Bakteriol. 274:118-125. Hughes, R., H. J. Olander, and C. B. Williams. 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res. 36:971-977. Itoh, K., T. Mitsuoka, K. Sudo, and K. Suzuki. 1983. Comparison of fecal flora of mice based upon different strains and different housing conditions. Z. Versuchstierkd. 25:135-146. Joens, L. A., and R. D. Glock. 1979. Experimental infection in mice with Treponema hyodysentenae. Infect. Immun. 25:757760. Joens, L. A., R. D. Glock, and J. M. Kinyon. 1980. Differentiation of Treponema hyodysenteriae from T. innocens by enteropathogenicity testing in the CF1 mouse. Vet. Rec. 107:527-529. Joens, L. A., I. M. Robinson, R. D. Glock, and P. J. Matthews. 1981. Production of lesions in gnotobiotic mice by inoculation with Treponema hyodysentenae. Infect. Immun. 31:504-506.

12a.Kennedy, M. J., K. R. Goodenough, C. P. Cornell, C. W. Ford, and R. J. Yancey, Jr. 1990. Abstr. Annu. Meet. Am. Soc.

Microbiol. 1990, p. 72. 13. Kunkle, R. A., and J. M. Kinyon. 1988. Improved selective

medium for the isolation of Treponema hyodysenteriae. J. Clin. Microbiol. 26:2357-2360. 14. Lemcke, R. M., and M. R. Burrows. 1982. Studies on a haemolysin produced by Treponema hyodysenteriae. J. Med. Microbiol. 15:205-214. 15. Meyer, R. C., J. Simon, and C. S. Byerly. 1975. The etiology of swine dysentery. III. The role of selected gram-negative obligate anaerobes. Vet. Pathol. 12:46-54. 16. Nibbelink, S. K. 1989. M.S. thesis, p. 105-115. Iowa State University, Ames.

17. Nibbelink, S. K., and M. J. Wannemuehler. 1990. Effect of Treponema hyodysenteriae infection on mucosal mast cells and T cells in the murine cecum. Infect. Immun. 58:88-92. 18. Nibbelink, S. K., and M. J. Wannemuehler. 1991. Susceptibility of inbred mouse strains to infection with Serpula (Treponema)

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hyodysenteriae. Infect. Immun. 59:3111-3118. 19. Nuessen, M. E., J. R. Birmingham, and L. A. Joens. 1982. Biological activity of a lipopolysaccharide extracted from Treponema hyodysenteriae. Infect. Immun. 37:138-142. 20. Nuessen, M. E., L. A. Joens, and R. D. Glock. 1983. Involvement of lipopolysaccharide in the pathogenicity of Treponema hyodysenteriae. J. Immunol. 131:997-999. 21. Saheb, S. A., L. Massicotte, and B. Picard. 1980. Purification and characterization of Treponema hyodysenteriae hemolysin. Bio-

INFECr. IMMUN.

chimie 62:779-785. 22. Suenaga, I., and T. Yamazaki. 1984. Experimental Treponema hyodysenteriae infection of mice. Zentralbl. Bakteriol. Mikrobiol. Hyg. Orig. Reihe A 257:348-356. 23. Whipp, S. C., I. M. Robinson, D. L. Harris, R. D. Glock, P. J. Matthews, and T. J. L. Alexander. 1979. Pathogenic synergism between Treponema hyodysenteriae and other selected anaerobes in gnotobiotic pigs. Infect. Immun. 26:1042-1047.