INFECGION AND IMMUNITY, Aug. 1992, p. 3117-3121 0019-9567/92/083117-05$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 60, No. 8
Comparison of Susceptibility of Inbred and Outbred Infant Mice to Escherichia coli Heat-Stable Enterotoxin STa ANNE BERTIN
Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Centre de Recherches de Tours, 37380 Nouzilly, France Received 24 March 1992/Accepted 4 May 1992
Comparison of the susceptibility of outbred OF1 and inbred BALB/c, C57BL/6, DBA/2, and CBA mice to heat-stable toxin (STa) of enterotoxigenic Escherichia coli was made at different levels of induced secretion. STa was able to elicit fluid accumulation into the intestine of each strain of mice; however, quantitatively different results were obtained. Results were as usual expressed by gut weight/remaining body weight ratios. Fluid accumulation weight and fluid accumulation weight/remaining body weight ratios were also estimated. Values obtained for BALB/c and OF1 mice were never significantly different, but values for OF1 mice were significantly higher than those for DBA and C57BV6 mice at the highest concentrations of toxin (toxin dilutions of 1/2, 1/4, and 1/5). At the highest toxin concentration, gut weight/remaining body weight ratio in C57BV6 mice was significantly lower than that for every other strain, but the fluid accumulation value obtained for DBA mice did not differ from that for C57BV6 mice. Fluid accumulation values for DBA mice were also significantly lower at toxin dilutions of 1/5 and 1/8 than those for every other strain, and this was also the case when estimating the fluid accumulation weight/remaining body weight ratio at a dilution of 1/8. Although the intestine of each strain of mice was able to respond to STa by fluid accumulation, differences in susceptibility of the STa receptor could exist and make DBA mice more resistant to enterotoxigenic E. coli diarrhea. understand the origin of these differences, I further investigated the susceptibility of the same strains of mice to STa. (Part of this work was presented as an abstract at the meeting "Genetique de l'hote et resistance aux maladies infectieuses et parasitaires. Bilan de l'action incitative programmee," 5 April 1990, Maison-Alfort, France [2a].)
Virulence factors have been demonstrated in enterotoxigenic Escherichia coli (ETEC) (5, 8, 10, 13). These are colonization factors (adhesins), often identified as piluslike structures, which favor the proliferation of bacteria in the intestine and toxins which stimulate the secretion of water and electrolytes (5, 8, 10, 13). Adhesins exhibit some species specificity; for instance, different CFA (colonization factor antigens) have been found in ETEC strains of human origin, K99 and F41 have been found in ETEC strains of bovine, porcine, and lamb origin, and K88 and 987P have been found in ETEC strains of porcine origin (5, 10, 13). Adhesins have also been found to be both plasmid and chromosome encoded, and although the adhesins are structurally and antigenically distinct, some genetic homology has been found in nonstructural genes for some adhesins (14, 15). Several plasmid-encoded toxins have also been identified (8). Heatlabile toxin has structural, functional, and antigenic similarities to cholera toxin (16). Heat-stable toxin (STa) has been shown to be active on the suckling mouse intestine (minor structural and genetic differences have been found for STa of human and porcine origin), and STh is only active in the pig intestine (26). Resistance to ETEC K88+ diarrheal disease in some pigs has been shown to be correlated to the nonadherence of bacteria to the brush borders of intestinal cells, and the genetics of the system have been analyzed (12, 20, 22, 23). However, differences in susceptibility were also found for other breeds of pig and with ETEC strains bearing other adhesins, and correlation between in vitro adherence of bacteria to intestinal cells and virulence was not always verified (3, 7). Recently, we tested possible differences in the susceptibility of inbred and outbred infant mice to ETEC strains bearing different adhesins (CFAI, CFAII, K88, K99, F41, 987P) and found differences for the various bacterial strains inoculated in one strain of mice, as well as for one bacterial strain inoculated in different strains of mice (6). To better
MATERIALS AND METHODS Animals. Swiss (outbred) OF1 mice and inbred strains BALB/cBy (H-2d), C57BV6 (H-2b), DBA/2 (H-2d), and CBA (H-y2) were obtained from IFFA-Credo (Saint GermainL'Arbresle, France) and raised as described previously (6). Toxin production. A single batch of culture filtrate containing STa was prepared from the bovine ETEC strain B41 (K99+ F41+ STa+) (2, 24) in CAA-YE medium (9) as described previously (1) and preserved at -20°C until used. Suckling mouse assay. A suckling mouse assay (4, 11) was used with modification for oral inoculation (25) as previously described (1). Each animal was orally inoculated either with the toxin diluted in deionized water or with deionized water (1 drop of 2% Evans blue dye added per 2 ml) in a 0.05-ml volume by using a 25 5/8-gauge needle fitted with a plastic tube. Each experiment for one dilution of the same batch of toxin, preserved as described before, was done the same day with the different strains of mice. Immediately before the test, suckling mice (36 to 72 h old) were gathered and randomly distributed into three or four groups of four animals for each strain. Each group was inoculated at 3-min intervals, beginning with one group of each strain and continuing in the same sequence. Animals were kept at 26°C before and during the test and were killed with chloroform 2 h after inoculation. At that time, the weights of the four intestines and of the remaining bodies were recorded for each group. Statistical analysis of data. The Student t test was used to compare values of gut weight/remaining body weight ratios, 3117
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TABLE 1. Gut weight, remaining body weight (carcass), and gut weight/carcass weight ratio obtained from control animals for the different strains of micea Strain (n)
C57BL/6 (7) 140
BALB/C 120
100
140
E
0,
~~~~~C57BU/6
z ] 120 ~ ~
0.30 0.35 0.31 0.42 0.41
+ t t t t
0.03 0.03 0.04 0.02 0.02
(g)C
Carcass wt
6.8 6.6 6.2 7.2 7.6
t t t t t
0.4 0.4 0.7 0.4 0.4
Gut wt/carcass
w4C
0.044 0.053 0.050 0.059 0.054
t t t t t
0.005 0.001 0.001 0.003 0.002
a Gut weight: each value is significantly different (P s 0.01) from the others except C57BL/6 versus DBA/2, DBA/2 versus CBA, and OF1 versus BALB/c. Carcass weight: 0.01 < P < 0.05 for BALB/c versus CBA, CBA versus DBA/2, and BALB/c versus DBA/2; P < 0.01 for OF1 versus C57BL/6, OF1 versus CBA, OF1 versus DBA/2. Gut weight/carcass weight: each value is significantly different (P . 0.01) except OFI versus CBA. b Number of groups of four animals. c Mean value + standard error.
0
o
CBA (7) DBA/2 (8) BALB/c (6) OF1 (8)
Gut wt (gf
~
~
100 80
140 DBA/2 120 100
onI
8 10 6 l 2 4 Reciprocal dilution of toxin
FIG. 1. Toxin activity assessed as gut weight/remaining body standard errors are given for four weight ratio. Mean values groups of four animals except for C57BL/6 and BALB/c mice at a dilution of 1/10, for which three groups of four animals were used. Toxin dilution 1/2: 0.01 < P c 0.05 for C57BU6 versus CBA, C57BL/6 versus DBA/2, CBA versus OF1, and DBA/2 versus BALB/c; P c 0.01 for C57BV/6 versus BALB/c, C57BLI6 versus OF1, and DBA/2 versus OF1. Toxin dilution 1/4: 0.01 < P c 0.05 for C57BV6 versus DBA/2; P c 0.01 for C57BV6 versus BALB/c, C57BL/6 versus OF1, CBA versus OF1, and DBA/2 versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for C57BLI6 versus CBA and DBA/2 versus CBA; P < 0.01 for DBA/2 versus BALB/c, DBA/2 versus OF1, C57BL/6 versus BALB/c, and C57BL/6 versus OF1. Toxin dilution 1/8: 0.01 < P c 0.05 for C57BV6 versus CBA; P < 0.01 for DBA/2 versus OF1, DBA/2 versus CBA, DBA/2 versus BALB/c, C57BLV6 versus OF1, and C57BV6 versus BALB/c. Toxin dilution 1/10: 0.01 < P < 0.05 for DBA/2 versus BALB/c.
fluid accumulation, or fluid accumulation/remaining body weight ratios, after calculation of these for each preparation of toxin and for each group of animals.
RESULTS Toxin-induced fluid accumulation assessed by gut weight/ remaining body weight (carcass) ratio was observed for each strain of mice (Fig. 1 and Table 1, for comparison with data obtained from control animals). Significantly different values were obtained for different strains of mice and different
levels of induced toxin secretion except between BALB/c and OF1 mice and between CBA and BALB/c mice, for which values were never significantly different. For the highest quantity of toxin administered, the value obtained for OF1 mice was significantly higher than those obtained for CBA, DBA, and C57BL/6 mice and the value obtained for C57BL/6 mice was significantly lower than that for every other strain (Fig. 1). Graphs also exhibited different shapes, particularly for strain C57BL/6, for which the results were not dose dependent (Fig. 1). Significant differences in gut weight, carcass weight, and gut weight/carcass weight ratios were also found between the strains of mice without toxin (Table 1). This reflects uncorrelated differences in gut weight and carcass weight between the different strains of mice. For this reason, an estimation of the quantity of fluid accumulated in the intestines was made by subtracting an estimated value of gut weight without toxin (Fig. 2). Results show that values obtained for DBA mice were not significantly different from those for C57BV6 mice, which gave the lowest value at a toxin dilution of 1/2, and were significantly lower than those for every other strain at toxin dilutions of 1/5 and 1/8 (Fig. 2). In addition, for the gut weight/carcass weight ratio, OF1 and BALB/c mice gave results which were not significantly different; C57BL6 mice at a dilution of 1/2 gave results significantly lower than those for BALB/c, OF1, and CBA mice. Significantly different results were found between BALB/c and CBA mice at dilutions of 1/4 and 1/5 (Fig. 2). When values obtained for fluid accumulation were compared with remaining body weight known for each animal, it was apparent that there was no significant difference between OF1 and BALB/c mice at each toxin dilution; however, at a dilution of 1/8, values for DBA mice were significantly lower than those for every other strain within which values were not significantly different (Fig. 3). DISCUSSION STa from ETEC was found to elicit fluid accumulation in the intestines of Swiss OF1 and inbred C57BV/6, CBA, DBA/2, and BALB/c infant mice. However, values obtained for gut weight/remaining body weight ratios, for fluid accumulation, or for fluid accumulation/remaining body weight ratios were not identical for every strain of mice tested. After inoculation of live bacteria into infant mice, it was
VOL. 60, 1992
INBRED AND OUTBRED MICE AND E. COLI TOXIN STa OF1
600.
80-
500.
3119
OF1
60-
400.
40.
300. 200-
BALB/C 80 -
BALB/C
600 500S.
60
400
cm 0,
40.
300. a
20
C,,
0
80
CBA
600-
E 60
500 -
0,
E
400
0
300
C0
40 20
I C57BL/6
LL
C57BL/6
600:
0 00
500 400
10 -
300 -
r..
zuu
*|*
600]
DBA/2
80 -
DBA/2
60-
50040
400-
.
300200
6 8 10 Reciprocal dilution of toxin
0
2
4
FIG. 2. Toxin activity assessed by fluid weight accumulated into intestines. Mean values + standard errors are given for four groups of four animals except for C57BV6 and BALB/c at a dilution of 1/10, for which three groups of four animals were used. Fluid accumulation values were calculated by subtracting for each group of four animals an estimated value of their gut weight without toxin. These were calculated from the known individual values of the carcass weight of each animal (individual values not shown) either with a linear regression curve established between intestinal weight and carcass weight of control animals when those were well correlated (>95% for DBA/2 and CBA) or from the mean yield of intestinal weight to carcass weight obtained in Table 1 for the other strains. Toxin dilution 1/2: 0.01 < P < 0.05 for C57BV6 versus BALB/c; P ' 0.01 for C57BV6 versus CBA, C57BL/6 versus OF1, DBAI2 versus CBA, and DBA/2 versus OF1. Toxin dilution 1/4: 0.01 < P < 0.05 for C57BU6 versus BALB/c and CBA versus BALB/c; P c 0.01 for DBA/2 versus BALB/c, DBA/2 versus OF1, C57BLV6 versus OF1, and CBA versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for CBA versus BALB/c; P c 0.01 for DBA/2 versus C57BV6, DBA/2 versus CBA, DBA/2 versus BALB/c, DBA/2 versus OF1, C57BV6 versus BALB/c, C57BV6 versus OF1, and CBA versus OF1. Toxin dilution 1/8: 0.01 < P 5 0.05 for DBA/2 versus CBA; P ' 0.01 for DBA/2 versus BALB/c, DBA/2 versus C57BLJ6, and DBA/2 versus OF1. Toxin dilution 1/10: 0.01 < P c 0.05 for DBA/2 versus CBA; P c 0.01 for DBA/2 versus BALB/c.
found that OF1 and CBA mice were highly susceptible to one bovine K99+ ETEC strain, in contrast to DBA/2, BALB/cBy, and C57BV/6 mice, which were found to be resistant. In addition, OF1, C57BL/6, BALB/cBy, and CBA
-
0 2 4 6 8 10 Reciprocal dilution of toxin
FIG. 3. Toxin activity assessed as accumulated fluid weight/ remaining body weight ratio. Mean values standard errors are given for four groups of four animals except for C57BV6 and BALB/c at a dilution of 1/10, for which three groups of four animals were used. Toxin dilution 1/2: 0.01 < P < 0.05 for C57BL/6 versus DBA and DBA versus OF1; P < 0.01 for C57BL/6 versus OF1 and CBA versus OF1. Toxin dilution 1/4: 0.01 < P < 0.05 for DBA/2 versus OF1; P < 0.01 for CBA versus OF1 and C57BL/6 versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for C57BL/6 versus OF1 and DBA/2 versus BALB/c; P < 0.01 for DBA/2 versus C57BL/6 and DBA/2 versus OF1. Toxin dilution 1/8: 0.01 < P c 0.05 for DBA/2 versus OF1 and DBA/2 versus BALB/c; P c 0.01 for DBA/2 versus CBA and DBA/2 versus C57BL/6.
mice were also found to be highly susceptible to F41+ or F41+ K99+ strains, as opposed to DBA/2 mice, which were found to be resistant (6). An infant mouse test without living bacteria is presently used in detecting the biological activity of STa (4, 11). To test the hypothesis that different strains of mice differed in susceptibility to STa and to compare the results obtained with those obtained with live bacteria, I tested the response of the intestine to toxin at different levels of induced toxin secretion for the same strains of mice as used in inoculation experiments (6). Results showed that the strains of mice tested were able to respond to toxin by accumulating fluid in the intestine. However, some quantitative results were found to be significantly different. For instance, at the highest level of induced secretion, BALB/c and OF1 mice exhibited values of gut
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weight/remaining body weight ratios which were significantly higher than those for DBA/2 and C57BL/6 mice, and C57BL/6 mice exhibited significantly lower values than those for all other strains tested. Infant Swiss mice have usually been used to test STa activity. One comparison between Swiss mice and some inbred strains of mice (C3H/HeJ, AWySn, B1OA/jax, and C57BL/1OJ) was made with undiluted culture filtrates (17). There were clear-cut responses for Swiss and inbred mice that distinguished ST (now STa)- and non-ST-producing strains of E. coli, as well as quantitative differences of susceptibility between the inbred strains of mice. It was concluded that it was preferable to use outbred strains to screen new isolates of E. coli for ST enterotoxin production (17). In this study, other inbred strains were used for comparison of results obtained with live bacteria (6). Different toxin dilutions were used because the quantity of toxin which can be present in the intestine at any given moment when live bacteria are present cannot be known. In fact, characteristics of the intestinal STa receptor could be different, and consequently so could the activity threshold, maximum induced secretion, and dose-response effect. Intestinal products could also modulate STa availability at the level of these receptors. These reasons could explain the different dose effect responses obtained in this study. This could be important in the evolution of disease. A sealed adult model was established for evaluation of cholera enterotoxin (18, 19). In this model, animals with the H-2q or H-21 haplotype were less responsive to cholera enterotoxin than were animals with haplotype H-2" or H-2d; outbred (Swiss) strains of mice gave intermediate results (19). No correlation has been found between the susceptibility to live ETEC bacteria and H-2 complex of mice in the infant mouse diarrhea model (6). From data presented here
concerning the susceptibility to STa, it was also found that thL.re was no correlation with the H-2 complex, since BALB/c and DBA/2 mice, both H-2d, exhibited significant differences in gut weight/carcass weight ratios at dilutions of 1/2, 1/5, 1/8, and 1/10; in accumulated fluid weight at dilutions of 1/5, 1/8, and 1/10; and in fluid weight/carcass weight ratios at dilutions of 1/5 and 1/8 (Fig. 1 to 3). Also, H-2c mice (CBA) did not give lower values than H-2" (C57BLV6) or H-2Y (DBA/2 and BALB/c) mice as they did with cholera enterotoxin in the sealed adult model (19). The gut weight/carcass weight ratio is normally used to measure STa activity. However, fluid loss or the fluid weight/carcass weight ratio could better reflect the dehydration effect since, as evidenced by the results obtained here, intestinal weight and carcass weight are not necessarily correlated in the different strains of mice. It has been suggested that some pigs (one litter in the study) are resistant to diarrheal disease because they lack a specific STa receptor in their intestinal epithelial cells (21). Results obtained here concerning toxin activity in infant mouse intestines do not suggest a similar lack of receptor, since fluid accumulation can be obtained in each strain of mice and particularly in DBA/2 mice, which were found to be resistant to every ETEC strain (6). However, this strain gave lower gut weight/carcass weight ratios at the lowest concentrations of toxin (significant at dilutions of 1/5 and 1/8) than CBA, BALB/c, and OF1 mice (Table 1), and when comparing fluid accumulation in the intestine, DBA/2 mice gave lower results than every other strain tested at the lowest toxin concentrations (significant at 1/5 and 1/8 dilutions).
INFECT. IMMUN.
ACKNOWLEDGMENTS This work was supported by grant 4835, "Genetique de l'h6te et resistance aux maladies infectieuses et parasitaires," from Institut National de la Recherche Agronomique. I am grateful to H. Le Roux and E. Rabouan for production and care of experimental animals. REFERENCES 1. Bertin, A. 1982. Effect of propranolol on the secretory activity of Escherichia coli heat-stable enterotoxin in the suckling mouse assay. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. A 251:522-528. 2. Bertin, A. 1985. F41 antigen as a virulence factor in the infant mouse model of Escherichia coli diarrhoea. J. Gen. Microbiol. 131:3037-3045. 2a.Bertin, A. 1990. Sensibilite comparee de diff6rentes lignees de souris a la secretion intestinale induite par la toxine STa des colibacilles enterotoxinogenes. Ann. Rech. Vet. 21:295-296. Ann. Rech. Vet. 21:291-308. 3. Bertin, A. M., and M. F. Duchet-Suchaux. 1991. Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European Large White pigs. Am. J. Vet. Res. 52:45-49. 4. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 5. De Graaf, F. K., and F. R. Mooi. 1986. The fimbrial adhesins of Escherichia coli. Adv. Microb. Physiol. 28:65-143. 6. Duchet-Suchaux, M., C. Le Maitre, and A. Bertin. 1990. Differences in susceptibility of inbred and outbred infant mice to enterotoxigenic Escherichia coli of bovine, porcine and human origin. J. Med. Microbiol. 31:185-190. 7. Duchet-Suchaux, M. F., A. M. Bertin, and P. S. Menanteau. 1991. Susceptibility of Chinese Meishan and European Large White pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41. Am. J. Vet. Res. 52:40-44. 8. Elwell, L. P. 1980. Plasmid-mediated factors associated with virulence of bacteria to animals. Annu. Rev. Microbiol. 34:465496. 9. Evans, D. G., D. J. Evans, and S. L. Gorbach. 1973. Identification of enterotoxigenic Eschenichia coli activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. 10. Gaastra, W., and F. K. De Graaf. 1982. Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol. Rev. 46:129-161. 11. Gianella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. 12. Gibbons, R. A., R. Selhwood, M. R. Burrows, and P. A. Hunter. 1977. Inherence of resistance to neonatal Eschenichia coli diarrhoea in the pig: examination of the genetic system. Theor. Appl. Genet. 51:65-70. 13. Klemm, P. 1985. Fimbrial adhesins of Escherichia coli. Rev. Infect. Dis. 7:321-340. 14. Korth, M. J., R. A. Schneider, and S. L. Moseley. 1991. An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells. Infect. Immun. 59:2333-2340. 15. Krogfelt, K. A. 1991. Bacterial adhesion: genetics, biogenesis, and role in pathogenesis of fimbrial adhesins of Escherichia coli. Rev. Infect. Dis. 13:721-735. 16. Moss, J., and M. Vaughan. 1980. Mechanism of activation of adenylate cyclase by choleragen and E. coli heat-labile enterotoxin, p. 107-126. In M. Field, J. S. Fordtran, and S. G. Schultz (ed.), Secretory diarrhea. Clinical physiology series. American Physiological Society, Bethesda, Md. 17. Pestana De Castro, A. F., M. B. Serafim, H. A. Rangel, and R. L. Guerrant. 1978. Swiss and inbred mice in the infant mouse test for the assay of Escherichia coli thermostable enterotoxin. Infect. Immun. 22:972-974.
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18. Richardson, S. H., J. C. Giles, and K. S. Kruger. 1984. Sealed adult mice: new model for enterotoxin evaluation. Infect. Immun. 43:482-486. 19. Richardson, S. H., and R. E. Kuhn. 1986. Studies on the genetic and cellular control of sensitivity to enterotoxins in the sealed adult mouse model. Infect. Immun. 54:522-528. 20. Rutter, J. M., M. R. Burrows, R. Sellwood, and R. A. Gibbons. 1975. A genetic basis for resistance to enteric disease caused by E. coli. Nature (London) 257:135-136. 21. Saeed, A. M., R. McMillian, V. Huckelberry, R. Abernathy, and R. N. Greenberg. 1987. Specific receptor for Eschenchia coli heat-stable enterotoxin (STa) may determine susceptibility of piglets to diarrheal disease. FEMS Microbiol. Lett. 43:247-251. 22. Seliwood, R. 1979. Eschenchia coli diarrhoea in pigs with or
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without the K88 receptor. Vet. Rec. 105:228-230. 23. Sellwood, R., R. A. Gibbons, G. W. Jones, and J. H. Rutter. 1975. Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: the existence of two pig phenotypes. J. Med. Microbiol. 8:405-411. 24. Smith, H. W. 1971. The bacteriology of the alimentary tract of domestic animals suffering from Escherichia coli infection. Ann. N.Y. Acad. Sci. 176:110-125. 25. Stavric, S., and D. Jeffrey. 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol. 23:331-336. 26. Thomson, M. R. 1987. Escherichia coli heat-stable enterotoxins and their receptors. Pathol. Immunopathol. Res. 6:103-116.
Vol. 60, No. 8
Comparison of Susceptibility of Inbred and Outbred Infant Mice to Escherichia coli Heat-Stable Enterotoxin STa ANNE BERTIN
Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Centre de Recherches de Tours, 37380 Nouzilly, France Received 24 March 1992/Accepted 4 May 1992
Comparison of the susceptibility of outbred OF1 and inbred BALB/c, C57BL/6, DBA/2, and CBA mice to heat-stable toxin (STa) of enterotoxigenic Escherichia coli was made at different levels of induced secretion. STa was able to elicit fluid accumulation into the intestine of each strain of mice; however, quantitatively different results were obtained. Results were as usual expressed by gut weight/remaining body weight ratios. Fluid accumulation weight and fluid accumulation weight/remaining body weight ratios were also estimated. Values obtained for BALB/c and OF1 mice were never significantly different, but values for OF1 mice were significantly higher than those for DBA and C57BV6 mice at the highest concentrations of toxin (toxin dilutions of 1/2, 1/4, and 1/5). At the highest toxin concentration, gut weight/remaining body weight ratio in C57BV6 mice was significantly lower than that for every other strain, but the fluid accumulation value obtained for DBA mice did not differ from that for C57BV6 mice. Fluid accumulation values for DBA mice were also significantly lower at toxin dilutions of 1/5 and 1/8 than those for every other strain, and this was also the case when estimating the fluid accumulation weight/remaining body weight ratio at a dilution of 1/8. Although the intestine of each strain of mice was able to respond to STa by fluid accumulation, differences in susceptibility of the STa receptor could exist and make DBA mice more resistant to enterotoxigenic E. coli diarrhea. understand the origin of these differences, I further investigated the susceptibility of the same strains of mice to STa. (Part of this work was presented as an abstract at the meeting "Genetique de l'hote et resistance aux maladies infectieuses et parasitaires. Bilan de l'action incitative programmee," 5 April 1990, Maison-Alfort, France [2a].)
Virulence factors have been demonstrated in enterotoxigenic Escherichia coli (ETEC) (5, 8, 10, 13). These are colonization factors (adhesins), often identified as piluslike structures, which favor the proliferation of bacteria in the intestine and toxins which stimulate the secretion of water and electrolytes (5, 8, 10, 13). Adhesins exhibit some species specificity; for instance, different CFA (colonization factor antigens) have been found in ETEC strains of human origin, K99 and F41 have been found in ETEC strains of bovine, porcine, and lamb origin, and K88 and 987P have been found in ETEC strains of porcine origin (5, 10, 13). Adhesins have also been found to be both plasmid and chromosome encoded, and although the adhesins are structurally and antigenically distinct, some genetic homology has been found in nonstructural genes for some adhesins (14, 15). Several plasmid-encoded toxins have also been identified (8). Heatlabile toxin has structural, functional, and antigenic similarities to cholera toxin (16). Heat-stable toxin (STa) has been shown to be active on the suckling mouse intestine (minor structural and genetic differences have been found for STa of human and porcine origin), and STh is only active in the pig intestine (26). Resistance to ETEC K88+ diarrheal disease in some pigs has been shown to be correlated to the nonadherence of bacteria to the brush borders of intestinal cells, and the genetics of the system have been analyzed (12, 20, 22, 23). However, differences in susceptibility were also found for other breeds of pig and with ETEC strains bearing other adhesins, and correlation between in vitro adherence of bacteria to intestinal cells and virulence was not always verified (3, 7). Recently, we tested possible differences in the susceptibility of inbred and outbred infant mice to ETEC strains bearing different adhesins (CFAI, CFAII, K88, K99, F41, 987P) and found differences for the various bacterial strains inoculated in one strain of mice, as well as for one bacterial strain inoculated in different strains of mice (6). To better
MATERIALS AND METHODS Animals. Swiss (outbred) OF1 mice and inbred strains BALB/cBy (H-2d), C57BV6 (H-2b), DBA/2 (H-2d), and CBA (H-y2) were obtained from IFFA-Credo (Saint GermainL'Arbresle, France) and raised as described previously (6). Toxin production. A single batch of culture filtrate containing STa was prepared from the bovine ETEC strain B41 (K99+ F41+ STa+) (2, 24) in CAA-YE medium (9) as described previously (1) and preserved at -20°C until used. Suckling mouse assay. A suckling mouse assay (4, 11) was used with modification for oral inoculation (25) as previously described (1). Each animal was orally inoculated either with the toxin diluted in deionized water or with deionized water (1 drop of 2% Evans blue dye added per 2 ml) in a 0.05-ml volume by using a 25 5/8-gauge needle fitted with a plastic tube. Each experiment for one dilution of the same batch of toxin, preserved as described before, was done the same day with the different strains of mice. Immediately before the test, suckling mice (36 to 72 h old) were gathered and randomly distributed into three or four groups of four animals for each strain. Each group was inoculated at 3-min intervals, beginning with one group of each strain and continuing in the same sequence. Animals were kept at 26°C before and during the test and were killed with chloroform 2 h after inoculation. At that time, the weights of the four intestines and of the remaining bodies were recorded for each group. Statistical analysis of data. The Student t test was used to compare values of gut weight/remaining body weight ratios, 3117
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INFECT. IMMUN.
BERTIN OF1
TABLE 1. Gut weight, remaining body weight (carcass), and gut weight/carcass weight ratio obtained from control animals for the different strains of micea Strain (n)
C57BL/6 (7) 140
BALB/C 120
100
140
E
0,
~~~~~C57BU/6
z ] 120 ~ ~
0.30 0.35 0.31 0.42 0.41
+ t t t t
0.03 0.03 0.04 0.02 0.02
(g)C
Carcass wt
6.8 6.6 6.2 7.2 7.6
t t t t t
0.4 0.4 0.7 0.4 0.4
Gut wt/carcass
w4C
0.044 0.053 0.050 0.059 0.054
t t t t t
0.005 0.001 0.001 0.003 0.002
a Gut weight: each value is significantly different (P s 0.01) from the others except C57BL/6 versus DBA/2, DBA/2 versus CBA, and OF1 versus BALB/c. Carcass weight: 0.01 < P < 0.05 for BALB/c versus CBA, CBA versus DBA/2, and BALB/c versus DBA/2; P < 0.01 for OF1 versus C57BL/6, OF1 versus CBA, OF1 versus DBA/2. Gut weight/carcass weight: each value is significantly different (P . 0.01) except OFI versus CBA. b Number of groups of four animals. c Mean value + standard error.
0
o
CBA (7) DBA/2 (8) BALB/c (6) OF1 (8)
Gut wt (gf
~
~
100 80
140 DBA/2 120 100
onI
8 10 6 l 2 4 Reciprocal dilution of toxin
FIG. 1. Toxin activity assessed as gut weight/remaining body standard errors are given for four weight ratio. Mean values groups of four animals except for C57BL/6 and BALB/c mice at a dilution of 1/10, for which three groups of four animals were used. Toxin dilution 1/2: 0.01 < P c 0.05 for C57BU6 versus CBA, C57BL/6 versus DBA/2, CBA versus OF1, and DBA/2 versus BALB/c; P c 0.01 for C57BV/6 versus BALB/c, C57BLI6 versus OF1, and DBA/2 versus OF1. Toxin dilution 1/4: 0.01 < P c 0.05 for C57BV6 versus DBA/2; P c 0.01 for C57BV6 versus BALB/c, C57BL/6 versus OF1, CBA versus OF1, and DBA/2 versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for C57BLI6 versus CBA and DBA/2 versus CBA; P < 0.01 for DBA/2 versus BALB/c, DBA/2 versus OF1, C57BL/6 versus BALB/c, and C57BL/6 versus OF1. Toxin dilution 1/8: 0.01 < P c 0.05 for C57BV6 versus CBA; P < 0.01 for DBA/2 versus OF1, DBA/2 versus CBA, DBA/2 versus BALB/c, C57BLV6 versus OF1, and C57BV6 versus BALB/c. Toxin dilution 1/10: 0.01 < P < 0.05 for DBA/2 versus BALB/c.
fluid accumulation, or fluid accumulation/remaining body weight ratios, after calculation of these for each preparation of toxin and for each group of animals.
RESULTS Toxin-induced fluid accumulation assessed by gut weight/ remaining body weight (carcass) ratio was observed for each strain of mice (Fig. 1 and Table 1, for comparison with data obtained from control animals). Significantly different values were obtained for different strains of mice and different
levels of induced toxin secretion except between BALB/c and OF1 mice and between CBA and BALB/c mice, for which values were never significantly different. For the highest quantity of toxin administered, the value obtained for OF1 mice was significantly higher than those obtained for CBA, DBA, and C57BL/6 mice and the value obtained for C57BL/6 mice was significantly lower than that for every other strain (Fig. 1). Graphs also exhibited different shapes, particularly for strain C57BL/6, for which the results were not dose dependent (Fig. 1). Significant differences in gut weight, carcass weight, and gut weight/carcass weight ratios were also found between the strains of mice without toxin (Table 1). This reflects uncorrelated differences in gut weight and carcass weight between the different strains of mice. For this reason, an estimation of the quantity of fluid accumulated in the intestines was made by subtracting an estimated value of gut weight without toxin (Fig. 2). Results show that values obtained for DBA mice were not significantly different from those for C57BV6 mice, which gave the lowest value at a toxin dilution of 1/2, and were significantly lower than those for every other strain at toxin dilutions of 1/5 and 1/8 (Fig. 2). In addition, for the gut weight/carcass weight ratio, OF1 and BALB/c mice gave results which were not significantly different; C57BL6 mice at a dilution of 1/2 gave results significantly lower than those for BALB/c, OF1, and CBA mice. Significantly different results were found between BALB/c and CBA mice at dilutions of 1/4 and 1/5 (Fig. 2). When values obtained for fluid accumulation were compared with remaining body weight known for each animal, it was apparent that there was no significant difference between OF1 and BALB/c mice at each toxin dilution; however, at a dilution of 1/8, values for DBA mice were significantly lower than those for every other strain within which values were not significantly different (Fig. 3). DISCUSSION STa from ETEC was found to elicit fluid accumulation in the intestines of Swiss OF1 and inbred C57BV/6, CBA, DBA/2, and BALB/c infant mice. However, values obtained for gut weight/remaining body weight ratios, for fluid accumulation, or for fluid accumulation/remaining body weight ratios were not identical for every strain of mice tested. After inoculation of live bacteria into infant mice, it was
VOL. 60, 1992
INBRED AND OUTBRED MICE AND E. COLI TOXIN STa OF1
600.
80-
500.
3119
OF1
60-
400.
40.
300. 200-
BALB/C 80 -
BALB/C
600 500S.
60
400
cm 0,
40.
300. a
20
C,,
0
80
CBA
600-
E 60
500 -
0,
E
400
0
300
C0
40 20
I C57BL/6
LL
C57BL/6
600:
0 00
500 400
10 -
300 -
r..
zuu
*|*
600]
DBA/2
80 -
DBA/2
60-
50040
400-
.
300200
6 8 10 Reciprocal dilution of toxin
0
2
4
FIG. 2. Toxin activity assessed by fluid weight accumulated into intestines. Mean values + standard errors are given for four groups of four animals except for C57BV6 and BALB/c at a dilution of 1/10, for which three groups of four animals were used. Fluid accumulation values were calculated by subtracting for each group of four animals an estimated value of their gut weight without toxin. These were calculated from the known individual values of the carcass weight of each animal (individual values not shown) either with a linear regression curve established between intestinal weight and carcass weight of control animals when those were well correlated (>95% for DBA/2 and CBA) or from the mean yield of intestinal weight to carcass weight obtained in Table 1 for the other strains. Toxin dilution 1/2: 0.01 < P < 0.05 for C57BV6 versus BALB/c; P ' 0.01 for C57BV6 versus CBA, C57BL/6 versus OF1, DBAI2 versus CBA, and DBA/2 versus OF1. Toxin dilution 1/4: 0.01 < P < 0.05 for C57BU6 versus BALB/c and CBA versus BALB/c; P c 0.01 for DBA/2 versus BALB/c, DBA/2 versus OF1, C57BLV6 versus OF1, and CBA versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for CBA versus BALB/c; P c 0.01 for DBA/2 versus C57BV6, DBA/2 versus CBA, DBA/2 versus BALB/c, DBA/2 versus OF1, C57BV6 versus BALB/c, C57BV6 versus OF1, and CBA versus OF1. Toxin dilution 1/8: 0.01 < P 5 0.05 for DBA/2 versus CBA; P ' 0.01 for DBA/2 versus BALB/c, DBA/2 versus C57BLJ6, and DBA/2 versus OF1. Toxin dilution 1/10: 0.01 < P c 0.05 for DBA/2 versus CBA; P c 0.01 for DBA/2 versus BALB/c.
found that OF1 and CBA mice were highly susceptible to one bovine K99+ ETEC strain, in contrast to DBA/2, BALB/cBy, and C57BV/6 mice, which were found to be resistant. In addition, OF1, C57BL/6, BALB/cBy, and CBA
-
0 2 4 6 8 10 Reciprocal dilution of toxin
FIG. 3. Toxin activity assessed as accumulated fluid weight/ remaining body weight ratio. Mean values standard errors are given for four groups of four animals except for C57BV6 and BALB/c at a dilution of 1/10, for which three groups of four animals were used. Toxin dilution 1/2: 0.01 < P < 0.05 for C57BL/6 versus DBA and DBA versus OF1; P < 0.01 for C57BL/6 versus OF1 and CBA versus OF1. Toxin dilution 1/4: 0.01 < P < 0.05 for DBA/2 versus OF1; P < 0.01 for CBA versus OF1 and C57BL/6 versus OF1. Toxin dilution 1/5: 0.01 < P c 0.05 for C57BL/6 versus OF1 and DBA/2 versus BALB/c; P < 0.01 for DBA/2 versus C57BL/6 and DBA/2 versus OF1. Toxin dilution 1/8: 0.01 < P c 0.05 for DBA/2 versus OF1 and DBA/2 versus BALB/c; P c 0.01 for DBA/2 versus CBA and DBA/2 versus C57BL/6.
mice were also found to be highly susceptible to F41+ or F41+ K99+ strains, as opposed to DBA/2 mice, which were found to be resistant (6). An infant mouse test without living bacteria is presently used in detecting the biological activity of STa (4, 11). To test the hypothesis that different strains of mice differed in susceptibility to STa and to compare the results obtained with those obtained with live bacteria, I tested the response of the intestine to toxin at different levels of induced toxin secretion for the same strains of mice as used in inoculation experiments (6). Results showed that the strains of mice tested were able to respond to toxin by accumulating fluid in the intestine. However, some quantitative results were found to be significantly different. For instance, at the highest level of induced secretion, BALB/c and OF1 mice exhibited values of gut
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BERTIN
weight/remaining body weight ratios which were significantly higher than those for DBA/2 and C57BL/6 mice, and C57BL/6 mice exhibited significantly lower values than those for all other strains tested. Infant Swiss mice have usually been used to test STa activity. One comparison between Swiss mice and some inbred strains of mice (C3H/HeJ, AWySn, B1OA/jax, and C57BL/1OJ) was made with undiluted culture filtrates (17). There were clear-cut responses for Swiss and inbred mice that distinguished ST (now STa)- and non-ST-producing strains of E. coli, as well as quantitative differences of susceptibility between the inbred strains of mice. It was concluded that it was preferable to use outbred strains to screen new isolates of E. coli for ST enterotoxin production (17). In this study, other inbred strains were used for comparison of results obtained with live bacteria (6). Different toxin dilutions were used because the quantity of toxin which can be present in the intestine at any given moment when live bacteria are present cannot be known. In fact, characteristics of the intestinal STa receptor could be different, and consequently so could the activity threshold, maximum induced secretion, and dose-response effect. Intestinal products could also modulate STa availability at the level of these receptors. These reasons could explain the different dose effect responses obtained in this study. This could be important in the evolution of disease. A sealed adult model was established for evaluation of cholera enterotoxin (18, 19). In this model, animals with the H-2q or H-21 haplotype were less responsive to cholera enterotoxin than were animals with haplotype H-2" or H-2d; outbred (Swiss) strains of mice gave intermediate results (19). No correlation has been found between the susceptibility to live ETEC bacteria and H-2 complex of mice in the infant mouse diarrhea model (6). From data presented here
concerning the susceptibility to STa, it was also found that thL.re was no correlation with the H-2 complex, since BALB/c and DBA/2 mice, both H-2d, exhibited significant differences in gut weight/carcass weight ratios at dilutions of 1/2, 1/5, 1/8, and 1/10; in accumulated fluid weight at dilutions of 1/5, 1/8, and 1/10; and in fluid weight/carcass weight ratios at dilutions of 1/5 and 1/8 (Fig. 1 to 3). Also, H-2c mice (CBA) did not give lower values than H-2" (C57BLV6) or H-2Y (DBA/2 and BALB/c) mice as they did with cholera enterotoxin in the sealed adult model (19). The gut weight/carcass weight ratio is normally used to measure STa activity. However, fluid loss or the fluid weight/carcass weight ratio could better reflect the dehydration effect since, as evidenced by the results obtained here, intestinal weight and carcass weight are not necessarily correlated in the different strains of mice. It has been suggested that some pigs (one litter in the study) are resistant to diarrheal disease because they lack a specific STa receptor in their intestinal epithelial cells (21). Results obtained here concerning toxin activity in infant mouse intestines do not suggest a similar lack of receptor, since fluid accumulation can be obtained in each strain of mice and particularly in DBA/2 mice, which were found to be resistant to every ETEC strain (6). However, this strain gave lower gut weight/carcass weight ratios at the lowest concentrations of toxin (significant at dilutions of 1/5 and 1/8) than CBA, BALB/c, and OF1 mice (Table 1), and when comparing fluid accumulation in the intestine, DBA/2 mice gave lower results than every other strain tested at the lowest toxin concentrations (significant at 1/5 and 1/8 dilutions).
INFECT. IMMUN.
ACKNOWLEDGMENTS This work was supported by grant 4835, "Genetique de l'h6te et resistance aux maladies infectieuses et parasitaires," from Institut National de la Recherche Agronomique. I am grateful to H. Le Roux and E. Rabouan for production and care of experimental animals. REFERENCES 1. Bertin, A. 1982. Effect of propranolol on the secretory activity of Escherichia coli heat-stable enterotoxin in the suckling mouse assay. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. A 251:522-528. 2. Bertin, A. 1985. F41 antigen as a virulence factor in the infant mouse model of Escherichia coli diarrhoea. J. Gen. Microbiol. 131:3037-3045. 2a.Bertin, A. 1990. Sensibilite comparee de diff6rentes lignees de souris a la secretion intestinale induite par la toxine STa des colibacilles enterotoxinogenes. Ann. Rech. Vet. 21:295-296. Ann. Rech. Vet. 21:291-308. 3. Bertin, A. M., and M. F. Duchet-Suchaux. 1991. Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European Large White pigs. Am. J. Vet. Res. 52:45-49. 4. Dean, A. G., Y. C. Ching, R. G. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 5. De Graaf, F. K., and F. R. Mooi. 1986. The fimbrial adhesins of Escherichia coli. Adv. Microb. Physiol. 28:65-143. 6. Duchet-Suchaux, M., C. Le Maitre, and A. Bertin. 1990. Differences in susceptibility of inbred and outbred infant mice to enterotoxigenic Escherichia coli of bovine, porcine and human origin. J. Med. Microbiol. 31:185-190. 7. Duchet-Suchaux, M. F., A. M. Bertin, and P. S. Menanteau. 1991. Susceptibility of Chinese Meishan and European Large White pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41. Am. J. Vet. Res. 52:40-44. 8. Elwell, L. P. 1980. Plasmid-mediated factors associated with virulence of bacteria to animals. Annu. Rev. Microbiol. 34:465496. 9. Evans, D. G., D. J. Evans, and S. L. Gorbach. 1973. Identification of enterotoxigenic Eschenichia coli activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. 10. Gaastra, W., and F. K. De Graaf. 1982. Host-specific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains. Microbiol. Rev. 46:129-161. 11. Gianella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. 12. Gibbons, R. A., R. Selhwood, M. R. Burrows, and P. A. Hunter. 1977. Inherence of resistance to neonatal Eschenichia coli diarrhoea in the pig: examination of the genetic system. Theor. Appl. Genet. 51:65-70. 13. Klemm, P. 1985. Fimbrial adhesins of Escherichia coli. Rev. Infect. Dis. 7:321-340. 14. Korth, M. J., R. A. Schneider, and S. L. Moseley. 1991. An F41-K88-related genetic determinant of bovine septicemic Escherichia coli mediates expression of CS31A fimbriae and adherence to epithelial cells. Infect. Immun. 59:2333-2340. 15. Krogfelt, K. A. 1991. Bacterial adhesion: genetics, biogenesis, and role in pathogenesis of fimbrial adhesins of Escherichia coli. Rev. Infect. Dis. 13:721-735. 16. Moss, J., and M. Vaughan. 1980. Mechanism of activation of adenylate cyclase by choleragen and E. coli heat-labile enterotoxin, p. 107-126. In M. Field, J. S. Fordtran, and S. G. Schultz (ed.), Secretory diarrhea. Clinical physiology series. American Physiological Society, Bethesda, Md. 17. Pestana De Castro, A. F., M. B. Serafim, H. A. Rangel, and R. L. Guerrant. 1978. Swiss and inbred mice in the infant mouse test for the assay of Escherichia coli thermostable enterotoxin. Infect. Immun. 22:972-974.
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18. Richardson, S. H., J. C. Giles, and K. S. Kruger. 1984. Sealed adult mice: new model for enterotoxin evaluation. Infect. Immun. 43:482-486. 19. Richardson, S. H., and R. E. Kuhn. 1986. Studies on the genetic and cellular control of sensitivity to enterotoxins in the sealed adult mouse model. Infect. Immun. 54:522-528. 20. Rutter, J. M., M. R. Burrows, R. Sellwood, and R. A. Gibbons. 1975. A genetic basis for resistance to enteric disease caused by E. coli. Nature (London) 257:135-136. 21. Saeed, A. M., R. McMillian, V. Huckelberry, R. Abernathy, and R. N. Greenberg. 1987. Specific receptor for Eschenchia coli heat-stable enterotoxin (STa) may determine susceptibility of piglets to diarrheal disease. FEMS Microbiol. Lett. 43:247-251. 22. Seliwood, R. 1979. Eschenchia coli diarrhoea in pigs with or
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without the K88 receptor. Vet. Rec. 105:228-230. 23. Sellwood, R., R. A. Gibbons, G. W. Jones, and J. H. Rutter. 1975. Adhesion of enteropathogenic Escherichia coli to pig intestinal brush borders: the existence of two pig phenotypes. J. Med. Microbiol. 8:405-411. 24. Smith, H. W. 1971. The bacteriology of the alimentary tract of domestic animals suffering from Escherichia coli infection. Ann. N.Y. Acad. Sci. 176:110-125. 25. Stavric, S., and D. Jeffrey. 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol. 23:331-336. 26. Thomson, M. R. 1987. Escherichia coli heat-stable enterotoxins and their receptors. Pathol. Immunopathol. Res. 6:103-116.
