Vol. 26, No. 3
INFECTION AND IMMUNITY, Dec. 1979, p. 1196-1201 0019-9567/79/12-1 196/06$02.00/0
Experimental Infection of Rabbit Ligated Heal Loops with Treponema hyodysenteriae FLOYD C. KNOOP Medical Department of Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178
Received for publication 18 September 1979
An in vivo animal model was used to assess the enteropathogenicity of the etiological agent (Treponema hyodysenteriae) of swine dysentery. Multiple ligated ileal loops, prepared in New Zealand white rabbits, were challenged with either pathogenic (B78 and B204) or nonpathogenic (Pu) isolates of the organism. The pathogenic isolates induced the onset of intestinal fluid accumulation as early as 4 h, with maximal fluid induction at 18 h postchallenge. Gross lesions of the intestinal mucosa, observed in ileal loops of rabbits sacrificed 24 h postchallenge, were characteristic of swine dysentery. Both pathogenic isolates colonized the epithelial surface and eroded the mucosal barrier, as determined by histological and scanning electron microscopic observations. Intestinal fluid accumulation and erosion of the mucosal barrier were not observed in ileal loops exposed to the nonpathogenic isolate (Pu) or to either of the nonviable pathogenic (B78 and B204) isolates. The ability of pathogenic isolates to initiate and produce infection in rabbit ligated ileal loops, which closely resembles the disease in swine, provides a system with which to study experimental swine dysentery.
Swine dysentery is a mucohemorrhagic diarrheal disease characterized by severe inflammation, excess mucus production, and necrosis of the large intestine (1, 4, 5, 14, 16). The etiological agent, Treponema hyodysenteriae, was isolated and characterized by Harris et al. (5). Although studies on the pathogenesis of the etiological agent have been primarily restricted to swine, several attempts to reproduce the disease in guinea pigs, mice, and rabbits, either uniformly or in a typical manner by parenteral injections of T. hyodysenteriae have failed (6). The oral challenge of germfree mice or of puppies harboring a normal flora with T. hyodysenteriae have failed to demonstrate the cardinal manifestations of the disease, the intestinal discharge of fluid and mucus accompanied by the appearance of hemorrhagic lesions in the intestinal mucosa (12). Whipp et al. (15) used multiple, ligated loops of swine to study the enteropathogenicity of T. hyodysenteriae. Although this in vivo model is relatively rapid and reliable, it has the disadvantage of size, cost, and laboratory adaptability for routine assay and largescale investigation. Recently, Joens et al. (7) developed an in vivo model by intragastric inoculation of guinea pigs with T. hyodysenteriae. A diarrheal syndrome was induced but did not parallel the hemorrhagic condition found in diseased swine. In the present investigation, experiments were performed that characterize the response of rab-
bit ligated ileal loops to infection with T. hyodysenteriae. This animal system should be useful to determine the sequence of events that culminate in swine dysentery. MATERLALS AND METHODS Microorganisms. Two pathogenic isolates, B78 and B204, and a nonpathogenic isolate, Pu, were obtained from J. M. Kinyon and D. L. Harris (College of Veterinary Medicine, Ames, Iowa). The enteropathogenicity of these isolates for swine has been determined (7, 12, 15). All stock cultures were maintained as described elsewhere (13). At the time of this investigation isolate Pu had been passaged 13 times on artificial media; isolates B78 and B204 had been passaged in excess of 50 times on artificial media. Growth medium. Trypticase soy broth without dextrose (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 10% fetal calf serum (GIBCO, Grand Island, N.Y.) was routinely used. The broth medium was prepared according to the aerobic method of Kinyon and Harris (11). Anaerobic culture was carried out by the method of Knoop et al. (13). Inocula. Broth cultures (24 h) of T. hyodysenteriae were transferred to sterile 50-ml conical centrifuge tubes, overlaid with an atmosphere of 70:30 deoxygenated H2-CO2 and centrifuged at 2,500 x g for 30 min. The resulting cell pellet was resuspended in fresh culture medium containing 200 ,g of spectinomycin (The Upjohn Co., Kalamazoo, Mich.) per ml to prevent normal bowel flora overgrowth and used immediately to challenge rabbit ligated ileal loops (see below). The inoculum was checked for contamination by phase contrast microscopy and by inoculation onto 5% sheep blood agar plates. Replicate blood plates were
1196
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INFECTION OF RABBITS WITH T. HYODYSENTERIAE
incubated aerobically at 37 and 230C, or anaerobically at 370C and checked after 4 to 6 days for contaminating microorganisms. Animals. New Zealand white rabbits (1.5 kg) were obtained from a commercial source (Daubert Rabbitry, Omaha, Nebr.) and housed in stainless steel isolation cages with wire grid floors to prevent copraphagic activity. All animals were maintained on Purina chow (Ralston Purina Co., St. Louis, Mo.); drugs were not added. Healthy animals without detectable intestinal malfunction were used for all investigative procedures; no preference was given to sex. The animals were deprived of food 24 h before surgery.
Surgical procedures. The ileal loop assay procedure was essentially that of Kasai and Burrows (8). The animals were anesthetized by ether, and a laparotomy was performed in the left abdominal wall approximately 2 cm from the mid-ventral line. The ileum and appendix were exposed and carefully moved to a peripheral gauze drape. At a distance of 100 cm anterior to the appendix, the ileum was ligated, and 10 ml of 0.067 M phosphate-buffered saline (pH 7.0) containing 200 jig of spectinomycin (Upjohn) per ml was injected into the intestinal lumen. The solution was moved toward the appendix by manual manipulation, and the ileum was ligated at a point 10 cm anterior to the appendix. Beginning at this point and progressing anteriorly, a series of six ileal loops (8 to 10 cm in length) were ligated with silk sutures and injected with 2.0 ml of inoculum via a syringe and 26gauge needle. The injection site was then separated from the intestinal loop by ligature. Care was taken not to disrupt the mesenteric membranes or vasculature. A solution consisting of 0.067 M phosphatebuffered saline (pH 7.0) supplemented with 200 tg of spectinomycin per ml was routinely used as a spray to prevent extraluminal dehydration. After challenge, the abdominal contents were replaced, the abdominal wall was sutured, and the outer muscle and skin layers were secured by 18-mm wound clips. The animals were returned to isolation cages and allowed water ad libitum. After 18 to 24 h the animals were sacrificed. Data collection. Postmortem examinations 18 to 24 h after challenge were performed. The ligated portion of the intestine was removed and placed on moistened filter paper. The individual loops were examined, and the amount of intraluminal fluid and length of each loop were recorded. Intestinal scrapings of intraluminal mucus were prepared for analysis by light microscopy. In further studies, each intestinal loop was placed in a sterile stainless steel Eberbach homogenizing vessel containing 25 ml of precooled (40C) fetal calf serum. The walls of the homogenizing vessel were sprayed with dimethylpolysiloxane, and a small crystal of n-octadecyl alcohol was added to reduce foaming. The vessel contents were then homogenized at 20,000 rpm for 10 s. A 1-ml amount of the homogenate was aseptically transferred to a sterile dilution blank containing 9 ml of fetal calf serum for viable count determinations. Standard 10-fold serial dilutions were performed. Viable counts were determined on duplicate 5% sheep blood agar plates after incubation at 370C for 4 to 6 days in an anaerobic GasPak jar. Atmospheric conditions were produced by GasPak (BBL) hydrogen and carbon dioxide generator enve-
1197
lopes. To determine the effect of the homogenization process on the viability of T. hyodysenteriae, 10 ml of a 24-h broth culture of each isolate was separately mixed with 25 ml of fetal calf serum. Standard dilution and plate counts were carried out before and after the homogenization process. The recovery of viable organisms per milliliter of sample was then determined. To examine histopathology and electrolyte concentrations, challenged animals were lightly anesthetized with ether, and the abdominal cavity and ligated ileum were exposed. The fluid from positive leal loops was immediately withdrawn by a syringe and an 18-gauge needle and replaced with 10% buffered Formalin. The loop was then excised, and 1-cm sections were prepared in the presence of fixative. After overnight fixation, routine histological sections were prepared, stained with hematoxylin-eosin, and examined by light microscopy. Specimens for scanning electron microscopy were prepared according to previous methods (13). The concentration of electrolytes in luminal fluid was determined in an ASTRA-8 Analyzer (Beckman Instruments, Inc., Fullerton, Calif.).
RESULTS Gross and light microscopic observations. Viable broth cultures of pathogenic T. hyodysenteriae isolates B78 and B204 induced gross hemorrhagic lesions of the intestinal mucosa and fluid accumulation in rabbit ligated ileal loops 18 to 24 h postchallenge. Varying degrees of cytopathology were observed, ranging from acute inflammation to discrete lesions. The lesions were petechial and contained numerous treponemes as evidenced by light microscopy of mucosal scrapings from the affected area (Fig. 1). The entire intestinal mucosa showed swelling, inflammation, and the presence of a pseudomembrane (Fig. 2A). The epithelium was flattened and partially or completely denuded of villi. In contrast, cultures of the nonpathogenic isolate (Pu) did not induce gross lesions or fluid accumulation (Fig. 2B). Intestinal fluid accumulation. The accumulation of intestinal fluid in ileal loops challenged with the pathogenic isolates (B78 and B204) began to occur 4 h postchallenge and reached a maximum 18 h after inoculation (Fig. 3). Harvested fluid contained (per liter): sodium (155 meq), potassium (6.5 meq), chloride (75 meq), and carbonate (68 meq). The fluid also contained a high content of mucus, often mixed with blood. This observation was found to be related to inoculum size. An inoculum of 3.5 x 107 colony-forming units or greater of isolate B204 would consistently result in mucus-containing fluid (Table 1). However, an inoculum of approximately 3.5 x 108 of isolate B78 was required for the induction of fluid. Although a lower inoculum would induce fluid accumulation, blood and mucus, and in some instances
1 198
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FIG. 1. Silver-stained mucosal smear from hemorrhagic area of rabbit intestine. Note the abundance of treponemal forms (arrows).
mucosal lesions, were not always apparent upon gross examination. The nonpathogenic isolate (Pu) did not induce fluid accumulation (Table 1). When the pathogenic isolates were heattreated at 560C for 60 min or held at 230C for several days, resulting in the loss of viability as evidenced by standard plate counts, fluid induction was not observed. Scanning electron microscopic observations. Representative photomicrographs of ileal loops inoculated with the pathogenic and nonpathogenic isolates are presented in Fig. 4. Spirochetes were observed on the mucosal surface and within the crypts of ileal loops challenged with the pathogenic isolates. The ileum, challenged with the pathogenic isolates (B78 and B204), showed the absence of a mucus barrier, although fibrin masses and pseudomembranous exudates were observed. The infection and destruction of villar tips was also observed (Fig. 4A and B), and treponemes were often present on the mucosal surface. In contrast, the inoculation of ileal loops with the nonpathogenic isolate (Pu) did not result in disruption of the mucus barrier or lead to the destruction of villar tips (Fig. 4C and D). DISCUSSION These studies characterize the reproducible response of rabbit ligated ileal loops to challenge with pathogenic isolates of T. hyodysenteriae. Fluid accumulation, mucus production, and hemorrhagic lesions consistent with swine dysentery were induced. Histological examination
of the intestinal mucosa of leal loops challenged with the pathogenic isolates revealed a strong inflammatory response, erosion of the superficial lamina propria, and the presence of a pseudomembranous exudate. These results are consistent with other recent observations in swine (5, 6, 12, 15). Attempts to detect enterotoxic activity in crude unconcentrated culture filtrates, harvested 18 h after inoculation, were not successful. However, the onset of fluid accumulation occurred at 4 h postchallenge, reaching a maximum after 18 h, when viable pathogenic isolates were used as inoculum. The electrolyte concentration at 18 h postchallenge showed the presence of ions at levels consistent with the classical "rice-water" stool induced by Vibrio cholerae. These observations may suggest that the accumulation of intestinal fluid is mediated by the guanylate cyclase-cyclic GMP or Na +-K +-ATPase systems. Giannella et al. (2) have suggested an alternate mechanism, related to the adenylate cyclase-cyclic AMP system and mucosal invasion, in the pathogenesis of salmonella-mediated intestinal fluid secretion. Kennedy et al. (10), using scanning electron microscopy, showed the close association of large numbers of treponemes with the epithelial surface and the crypts in the colonic mucosa of swine. Glock et al. (3), by means of transmission electron microscopy, found similar results. However, large spirochetes were found intact within epithelial and goblet cells. In the present study, spirochetes with compatible T. hyodysenteriae
VOL. 26, 1979
INFECTION OF RABBITS WITH T. HYODYSENTERIAE11199
FIG. 2. Histological section of rabbit ileum 24 h after challenge with T. hyodysenteriae. Photomicrographs represent rabbit ileum challenged with isolates B204 (A) and Pu (B). Note the appearance of a pseudomembrane (pm) and swelling (sw) and the absence of intestinal villi (av) in (A). The presence of normal intestinal villi (nv) and absence of a pseudomembrane and swelling are also presented (B); control buffer gave similar results.
1200
INFECT. IMMUN.
KNOOP
morphology were associated with the mucosal bacterial forms, have been found in colonic lesurface and lesions of rabbit ligated ileal loops sions of swine intestine (9). Throughout the preswhen viewed by scanning electron microscopy. ent investigation, attempts were made to obFibrin masses, pseudomembranous exudates, and the destruction of intestinal villi and epithe- TABLE 1. Effect of T. hyodysenteriae infection on fluid accumulation in rabbit ligated ileal loops' lium were observed. Spirochetes, with variable numbers of other Inoculum Recovery at 24 Fluid accumuIsolate h postinfection lation ratio" (CFU)h 2
Pu
4.6 x 109
B78 B78 B78 B78
4.0 5.1 3.3 1.5
x 106 x 107 x 108 x 109
NDd
Negative
ND ND 2.9 x 109 2.3 x 109
Negative Negative 1.89 1.69
0
0
0
4
8
12
18
24
HOURS POST INFECTION
FIG. 3. Effect of T. hyodysenteriae on the accumulation of fluid in rabbit ligated ileal loops. The ratio represents milliliters of fluid accumulated per centimeter of intestinal loop; each point represents a mean value ± standard deviation from at least four separate experiments. See text for details of the experiment.
3.8 x 104 1.1 x 10 B204 Negative 3.8 x 105 2.3 x 102 B204 Negative 4.2 x 104 4.8 x 106 B204 Negative 1.2 x 109 1.73 B204 3.5 x 107 2.1 x 109 1.92 B204 4.5 x 108 B204 1.8 x 109 2.6 x 109 1.84 a Each figure represents the mean from three or more determinations. b CFU, Colony-forming units. 'Fluid accumulation ratios represent the mean intestinal fluid accumulation (milliliters) per centimeter of ligated ileal loop. d ND, Not determined.
FIG. 4. Scanning electron microscopy of rabbit ileum 24 h after challenge with T. hyodysenteriae. Photomicrographs represent the destruction of villi (A) and villar tips (B) by isolate B204; normal villi (C) and villar tips (D) were present after challenge with isolate Pu or control buffer.
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INFECTION OF RABBITS WITH T. HYODYSENTERIAE
serve and isolate other microflora that may contribute to the enteropathogenicity of T. hyodysenteriae. Numerous microscopic observations of mucosal scrapings and luminal fluid showed relatively few other detectable microorganisms. In addition, relatively few contaminating microorganisms were observed during viable count determinations. The role of spectinomycin in elimination of the normal flora and in the initiation of infection by T. hyodysenteriae was not determined. In summary, this animal system resembles the natural disease of swine and will be useful as a rapid and inexpensive model to study the process of infectivity, to assess enteropathogenicity, to elucidate the effectiveness of pharmacological agents, and to test the possible efficacy of vaccine products. ACKNOWLEDGMENT This work was supported by a grant from The National Pork Producers Council, Des Moines, Iowa. LITERATURE CITED 1. Alexander, T. J. L., and D. J. Taylor. 1969. The clinical signs, diagnosis, and control of swine dysentery. Vet. Rec. 85:59-63. 2. Giannella, R. A., R. E. Gots, A. N. Charney, W. B. Greenough, HI, and S. B. Formal. 1975. Pathogenesis of salmonella-mediated intestinal fluid secretion. Activation of adenylate cyclase and inhibition by indomethacin. Gasteroenterology 69:1238-1245. 3. Glock, R. D., D. L. Harris, and J. P. Kluge. 1974. Localization of spirochetes with the structural characteristics of Treponema hyodysenteriae in the lesions of swine dysentery. Infect. Immun. 9:167-178. 4. Harris, D. L., and R. D. Glock. 1972. Swine dysentery.
1201
Am. Vet. Med. Assoc. 160:561-565. 5. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon. 1972. Swine dysentery. I. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin. 67:61-64. 6. Hughes, R., H. J. Colander, and C. B. Williams. 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res. 36:971-978. 7. Joens, L. A., J. G. Songer, D. L. Harris, and R. D. Glock. 1978. Experimental infection with Treponema hyodysenteriae in guinea pigs. Infect. Immun. 22:132135. 8. Kasai, G. J., and W. Burrows. 1966. The titration of cholera toxin and antitoxin in the rabbit ileal loop. J. Infect. Dis. 116:606-614. 9. Kennedy, G. A., and A. C. Strafuss. 1976. Scanning electron microscopy of the lesions of swine dysentery. Am. J. Vet. Res. 37:395-401. 10. Kennedy, G. A., A. C. Strafuss, and D. A. Schoneweis. 1973. Scanning electron microscopic observations on swine dysentery. J. Am. Vet. Med. Assoc. 163:5355. 11. Kinyon, J. M., and D. L. Harris. 1974. Growth of Treponema hyodysenteriae in liquid medium. Vet. Rec. 95: 219-220. 12. Kinyon, J. M., D. L. Harris, and R. D. Glock. 1977. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immun. 15:638-646. 13. Knoop, F. C., G. D. Schrank, and F. M. Ferraro. 1979. In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells. Can. J. Microbiol. 25:399405. 14. Lussier, G. 1962. Vibrionic dysentery of swine in Ontario. I. Clinical aspects of pathology. Can. Vet. J. 3:237-288. 15. Whipp, S. C., D. L. Harris, J. M. Kinyon, K. G. Songer, and R. D. Glock. 1978. Enteropathogenicity testing of Treponema hyodysenteriae in ligated colonic loops of swine. Am. J. Vet. Res. 39:1293-1296. 16. Whiting, R. A., L. P. Doyle, and R. S. Spray. 1921. Swine dysentery. Purdue Univ. Agric. Exp. Stn. Res. Bull. 257:3-15.
INFECTION AND IMMUNITY, Dec. 1979, p. 1196-1201 0019-9567/79/12-1 196/06$02.00/0
Experimental Infection of Rabbit Ligated Heal Loops with Treponema hyodysenteriae FLOYD C. KNOOP Medical Department of Microbiology, Creighton University School of Medicine, Omaha, Nebraska 68178
Received for publication 18 September 1979
An in vivo animal model was used to assess the enteropathogenicity of the etiological agent (Treponema hyodysenteriae) of swine dysentery. Multiple ligated ileal loops, prepared in New Zealand white rabbits, were challenged with either pathogenic (B78 and B204) or nonpathogenic (Pu) isolates of the organism. The pathogenic isolates induced the onset of intestinal fluid accumulation as early as 4 h, with maximal fluid induction at 18 h postchallenge. Gross lesions of the intestinal mucosa, observed in ileal loops of rabbits sacrificed 24 h postchallenge, were characteristic of swine dysentery. Both pathogenic isolates colonized the epithelial surface and eroded the mucosal barrier, as determined by histological and scanning electron microscopic observations. Intestinal fluid accumulation and erosion of the mucosal barrier were not observed in ileal loops exposed to the nonpathogenic isolate (Pu) or to either of the nonviable pathogenic (B78 and B204) isolates. The ability of pathogenic isolates to initiate and produce infection in rabbit ligated ileal loops, which closely resembles the disease in swine, provides a system with which to study experimental swine dysentery.
Swine dysentery is a mucohemorrhagic diarrheal disease characterized by severe inflammation, excess mucus production, and necrosis of the large intestine (1, 4, 5, 14, 16). The etiological agent, Treponema hyodysenteriae, was isolated and characterized by Harris et al. (5). Although studies on the pathogenesis of the etiological agent have been primarily restricted to swine, several attempts to reproduce the disease in guinea pigs, mice, and rabbits, either uniformly or in a typical manner by parenteral injections of T. hyodysenteriae have failed (6). The oral challenge of germfree mice or of puppies harboring a normal flora with T. hyodysenteriae have failed to demonstrate the cardinal manifestations of the disease, the intestinal discharge of fluid and mucus accompanied by the appearance of hemorrhagic lesions in the intestinal mucosa (12). Whipp et al. (15) used multiple, ligated loops of swine to study the enteropathogenicity of T. hyodysenteriae. Although this in vivo model is relatively rapid and reliable, it has the disadvantage of size, cost, and laboratory adaptability for routine assay and largescale investigation. Recently, Joens et al. (7) developed an in vivo model by intragastric inoculation of guinea pigs with T. hyodysenteriae. A diarrheal syndrome was induced but did not parallel the hemorrhagic condition found in diseased swine. In the present investigation, experiments were performed that characterize the response of rab-
bit ligated ileal loops to infection with T. hyodysenteriae. This animal system should be useful to determine the sequence of events that culminate in swine dysentery. MATERLALS AND METHODS Microorganisms. Two pathogenic isolates, B78 and B204, and a nonpathogenic isolate, Pu, were obtained from J. M. Kinyon and D. L. Harris (College of Veterinary Medicine, Ames, Iowa). The enteropathogenicity of these isolates for swine has been determined (7, 12, 15). All stock cultures were maintained as described elsewhere (13). At the time of this investigation isolate Pu had been passaged 13 times on artificial media; isolates B78 and B204 had been passaged in excess of 50 times on artificial media. Growth medium. Trypticase soy broth without dextrose (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 10% fetal calf serum (GIBCO, Grand Island, N.Y.) was routinely used. The broth medium was prepared according to the aerobic method of Kinyon and Harris (11). Anaerobic culture was carried out by the method of Knoop et al. (13). Inocula. Broth cultures (24 h) of T. hyodysenteriae were transferred to sterile 50-ml conical centrifuge tubes, overlaid with an atmosphere of 70:30 deoxygenated H2-CO2 and centrifuged at 2,500 x g for 30 min. The resulting cell pellet was resuspended in fresh culture medium containing 200 ,g of spectinomycin (The Upjohn Co., Kalamazoo, Mich.) per ml to prevent normal bowel flora overgrowth and used immediately to challenge rabbit ligated ileal loops (see below). The inoculum was checked for contamination by phase contrast microscopy and by inoculation onto 5% sheep blood agar plates. Replicate blood plates were
1196
VOL. 26, 1979
INFECTION OF RABBITS WITH T. HYODYSENTERIAE
incubated aerobically at 37 and 230C, or anaerobically at 370C and checked after 4 to 6 days for contaminating microorganisms. Animals. New Zealand white rabbits (1.5 kg) were obtained from a commercial source (Daubert Rabbitry, Omaha, Nebr.) and housed in stainless steel isolation cages with wire grid floors to prevent copraphagic activity. All animals were maintained on Purina chow (Ralston Purina Co., St. Louis, Mo.); drugs were not added. Healthy animals without detectable intestinal malfunction were used for all investigative procedures; no preference was given to sex. The animals were deprived of food 24 h before surgery.
Surgical procedures. The ileal loop assay procedure was essentially that of Kasai and Burrows (8). The animals were anesthetized by ether, and a laparotomy was performed in the left abdominal wall approximately 2 cm from the mid-ventral line. The ileum and appendix were exposed and carefully moved to a peripheral gauze drape. At a distance of 100 cm anterior to the appendix, the ileum was ligated, and 10 ml of 0.067 M phosphate-buffered saline (pH 7.0) containing 200 jig of spectinomycin (Upjohn) per ml was injected into the intestinal lumen. The solution was moved toward the appendix by manual manipulation, and the ileum was ligated at a point 10 cm anterior to the appendix. Beginning at this point and progressing anteriorly, a series of six ileal loops (8 to 10 cm in length) were ligated with silk sutures and injected with 2.0 ml of inoculum via a syringe and 26gauge needle. The injection site was then separated from the intestinal loop by ligature. Care was taken not to disrupt the mesenteric membranes or vasculature. A solution consisting of 0.067 M phosphatebuffered saline (pH 7.0) supplemented with 200 tg of spectinomycin per ml was routinely used as a spray to prevent extraluminal dehydration. After challenge, the abdominal contents were replaced, the abdominal wall was sutured, and the outer muscle and skin layers were secured by 18-mm wound clips. The animals were returned to isolation cages and allowed water ad libitum. After 18 to 24 h the animals were sacrificed. Data collection. Postmortem examinations 18 to 24 h after challenge were performed. The ligated portion of the intestine was removed and placed on moistened filter paper. The individual loops were examined, and the amount of intraluminal fluid and length of each loop were recorded. Intestinal scrapings of intraluminal mucus were prepared for analysis by light microscopy. In further studies, each intestinal loop was placed in a sterile stainless steel Eberbach homogenizing vessel containing 25 ml of precooled (40C) fetal calf serum. The walls of the homogenizing vessel were sprayed with dimethylpolysiloxane, and a small crystal of n-octadecyl alcohol was added to reduce foaming. The vessel contents were then homogenized at 20,000 rpm for 10 s. A 1-ml amount of the homogenate was aseptically transferred to a sterile dilution blank containing 9 ml of fetal calf serum for viable count determinations. Standard 10-fold serial dilutions were performed. Viable counts were determined on duplicate 5% sheep blood agar plates after incubation at 370C for 4 to 6 days in an anaerobic GasPak jar. Atmospheric conditions were produced by GasPak (BBL) hydrogen and carbon dioxide generator enve-
1197
lopes. To determine the effect of the homogenization process on the viability of T. hyodysenteriae, 10 ml of a 24-h broth culture of each isolate was separately mixed with 25 ml of fetal calf serum. Standard dilution and plate counts were carried out before and after the homogenization process. The recovery of viable organisms per milliliter of sample was then determined. To examine histopathology and electrolyte concentrations, challenged animals were lightly anesthetized with ether, and the abdominal cavity and ligated ileum were exposed. The fluid from positive leal loops was immediately withdrawn by a syringe and an 18-gauge needle and replaced with 10% buffered Formalin. The loop was then excised, and 1-cm sections were prepared in the presence of fixative. After overnight fixation, routine histological sections were prepared, stained with hematoxylin-eosin, and examined by light microscopy. Specimens for scanning electron microscopy were prepared according to previous methods (13). The concentration of electrolytes in luminal fluid was determined in an ASTRA-8 Analyzer (Beckman Instruments, Inc., Fullerton, Calif.).
RESULTS Gross and light microscopic observations. Viable broth cultures of pathogenic T. hyodysenteriae isolates B78 and B204 induced gross hemorrhagic lesions of the intestinal mucosa and fluid accumulation in rabbit ligated ileal loops 18 to 24 h postchallenge. Varying degrees of cytopathology were observed, ranging from acute inflammation to discrete lesions. The lesions were petechial and contained numerous treponemes as evidenced by light microscopy of mucosal scrapings from the affected area (Fig. 1). The entire intestinal mucosa showed swelling, inflammation, and the presence of a pseudomembrane (Fig. 2A). The epithelium was flattened and partially or completely denuded of villi. In contrast, cultures of the nonpathogenic isolate (Pu) did not induce gross lesions or fluid accumulation (Fig. 2B). Intestinal fluid accumulation. The accumulation of intestinal fluid in ileal loops challenged with the pathogenic isolates (B78 and B204) began to occur 4 h postchallenge and reached a maximum 18 h after inoculation (Fig. 3). Harvested fluid contained (per liter): sodium (155 meq), potassium (6.5 meq), chloride (75 meq), and carbonate (68 meq). The fluid also contained a high content of mucus, often mixed with blood. This observation was found to be related to inoculum size. An inoculum of 3.5 x 107 colony-forming units or greater of isolate B204 would consistently result in mucus-containing fluid (Table 1). However, an inoculum of approximately 3.5 x 108 of isolate B78 was required for the induction of fluid. Although a lower inoculum would induce fluid accumulation, blood and mucus, and in some instances
1 198
INFECT. IMMUN.
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FIG. 1. Silver-stained mucosal smear from hemorrhagic area of rabbit intestine. Note the abundance of treponemal forms (arrows).
mucosal lesions, were not always apparent upon gross examination. The nonpathogenic isolate (Pu) did not induce fluid accumulation (Table 1). When the pathogenic isolates were heattreated at 560C for 60 min or held at 230C for several days, resulting in the loss of viability as evidenced by standard plate counts, fluid induction was not observed. Scanning electron microscopic observations. Representative photomicrographs of ileal loops inoculated with the pathogenic and nonpathogenic isolates are presented in Fig. 4. Spirochetes were observed on the mucosal surface and within the crypts of ileal loops challenged with the pathogenic isolates. The ileum, challenged with the pathogenic isolates (B78 and B204), showed the absence of a mucus barrier, although fibrin masses and pseudomembranous exudates were observed. The infection and destruction of villar tips was also observed (Fig. 4A and B), and treponemes were often present on the mucosal surface. In contrast, the inoculation of ileal loops with the nonpathogenic isolate (Pu) did not result in disruption of the mucus barrier or lead to the destruction of villar tips (Fig. 4C and D). DISCUSSION These studies characterize the reproducible response of rabbit ligated ileal loops to challenge with pathogenic isolates of T. hyodysenteriae. Fluid accumulation, mucus production, and hemorrhagic lesions consistent with swine dysentery were induced. Histological examination
of the intestinal mucosa of leal loops challenged with the pathogenic isolates revealed a strong inflammatory response, erosion of the superficial lamina propria, and the presence of a pseudomembranous exudate. These results are consistent with other recent observations in swine (5, 6, 12, 15). Attempts to detect enterotoxic activity in crude unconcentrated culture filtrates, harvested 18 h after inoculation, were not successful. However, the onset of fluid accumulation occurred at 4 h postchallenge, reaching a maximum after 18 h, when viable pathogenic isolates were used as inoculum. The electrolyte concentration at 18 h postchallenge showed the presence of ions at levels consistent with the classical "rice-water" stool induced by Vibrio cholerae. These observations may suggest that the accumulation of intestinal fluid is mediated by the guanylate cyclase-cyclic GMP or Na +-K +-ATPase systems. Giannella et al. (2) have suggested an alternate mechanism, related to the adenylate cyclase-cyclic AMP system and mucosal invasion, in the pathogenesis of salmonella-mediated intestinal fluid secretion. Kennedy et al. (10), using scanning electron microscopy, showed the close association of large numbers of treponemes with the epithelial surface and the crypts in the colonic mucosa of swine. Glock et al. (3), by means of transmission electron microscopy, found similar results. However, large spirochetes were found intact within epithelial and goblet cells. In the present study, spirochetes with compatible T. hyodysenteriae
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INFECTION OF RABBITS WITH T. HYODYSENTERIAE11199
FIG. 2. Histological section of rabbit ileum 24 h after challenge with T. hyodysenteriae. Photomicrographs represent rabbit ileum challenged with isolates B204 (A) and Pu (B). Note the appearance of a pseudomembrane (pm) and swelling (sw) and the absence of intestinal villi (av) in (A). The presence of normal intestinal villi (nv) and absence of a pseudomembrane and swelling are also presented (B); control buffer gave similar results.
1200
INFECT. IMMUN.
KNOOP
morphology were associated with the mucosal bacterial forms, have been found in colonic lesurface and lesions of rabbit ligated ileal loops sions of swine intestine (9). Throughout the preswhen viewed by scanning electron microscopy. ent investigation, attempts were made to obFibrin masses, pseudomembranous exudates, and the destruction of intestinal villi and epithe- TABLE 1. Effect of T. hyodysenteriae infection on fluid accumulation in rabbit ligated ileal loops' lium were observed. Spirochetes, with variable numbers of other Inoculum Recovery at 24 Fluid accumuIsolate h postinfection lation ratio" (CFU)h 2
Pu
4.6 x 109
B78 B78 B78 B78
4.0 5.1 3.3 1.5
x 106 x 107 x 108 x 109
NDd
Negative
ND ND 2.9 x 109 2.3 x 109
Negative Negative 1.89 1.69
0
0
0
4
8
12
18
24
HOURS POST INFECTION
FIG. 3. Effect of T. hyodysenteriae on the accumulation of fluid in rabbit ligated ileal loops. The ratio represents milliliters of fluid accumulated per centimeter of intestinal loop; each point represents a mean value ± standard deviation from at least four separate experiments. See text for details of the experiment.
3.8 x 104 1.1 x 10 B204 Negative 3.8 x 105 2.3 x 102 B204 Negative 4.2 x 104 4.8 x 106 B204 Negative 1.2 x 109 1.73 B204 3.5 x 107 2.1 x 109 1.92 B204 4.5 x 108 B204 1.8 x 109 2.6 x 109 1.84 a Each figure represents the mean from three or more determinations. b CFU, Colony-forming units. 'Fluid accumulation ratios represent the mean intestinal fluid accumulation (milliliters) per centimeter of ligated ileal loop. d ND, Not determined.
FIG. 4. Scanning electron microscopy of rabbit ileum 24 h after challenge with T. hyodysenteriae. Photomicrographs represent the destruction of villi (A) and villar tips (B) by isolate B204; normal villi (C) and villar tips (D) were present after challenge with isolate Pu or control buffer.
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INFECTION OF RABBITS WITH T. HYODYSENTERIAE
serve and isolate other microflora that may contribute to the enteropathogenicity of T. hyodysenteriae. Numerous microscopic observations of mucosal scrapings and luminal fluid showed relatively few other detectable microorganisms. In addition, relatively few contaminating microorganisms were observed during viable count determinations. The role of spectinomycin in elimination of the normal flora and in the initiation of infection by T. hyodysenteriae was not determined. In summary, this animal system resembles the natural disease of swine and will be useful as a rapid and inexpensive model to study the process of infectivity, to assess enteropathogenicity, to elucidate the effectiveness of pharmacological agents, and to test the possible efficacy of vaccine products. ACKNOWLEDGMENT This work was supported by a grant from The National Pork Producers Council, Des Moines, Iowa. LITERATURE CITED 1. Alexander, T. J. L., and D. J. Taylor. 1969. The clinical signs, diagnosis, and control of swine dysentery. Vet. Rec. 85:59-63. 2. Giannella, R. A., R. E. Gots, A. N. Charney, W. B. Greenough, HI, and S. B. Formal. 1975. Pathogenesis of salmonella-mediated intestinal fluid secretion. Activation of adenylate cyclase and inhibition by indomethacin. Gasteroenterology 69:1238-1245. 3. Glock, R. D., D. L. Harris, and J. P. Kluge. 1974. Localization of spirochetes with the structural characteristics of Treponema hyodysenteriae in the lesions of swine dysentery. Infect. Immun. 9:167-178. 4. Harris, D. L., and R. D. Glock. 1972. Swine dysentery.
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Am. Vet. Med. Assoc. 160:561-565. 5. Harris, D. L., R. D. Glock, C. R. Christensen, and J. M. Kinyon. 1972. Swine dysentery. I. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet. Med. Small Anim. Clin. 67:61-64. 6. Hughes, R., H. J. Colander, and C. B. Williams. 1975. Swine dysentery: pathogenicity of Treponema hyodysenteriae. Am. J. Vet. Res. 36:971-978. 7. Joens, L. A., J. G. Songer, D. L. Harris, and R. D. Glock. 1978. Experimental infection with Treponema hyodysenteriae in guinea pigs. Infect. Immun. 22:132135. 8. Kasai, G. J., and W. Burrows. 1966. The titration of cholera toxin and antitoxin in the rabbit ileal loop. J. Infect. Dis. 116:606-614. 9. Kennedy, G. A., and A. C. Strafuss. 1976. Scanning electron microscopy of the lesions of swine dysentery. Am. J. Vet. Res. 37:395-401. 10. Kennedy, G. A., A. C. Strafuss, and D. A. Schoneweis. 1973. Scanning electron microscopic observations on swine dysentery. J. Am. Vet. Med. Assoc. 163:5355. 11. Kinyon, J. M., and D. L. Harris. 1974. Growth of Treponema hyodysenteriae in liquid medium. Vet. Rec. 95: 219-220. 12. Kinyon, J. M., D. L. Harris, and R. D. Glock. 1977. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immun. 15:638-646. 13. Knoop, F. C., G. D. Schrank, and F. M. Ferraro. 1979. In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells. Can. J. Microbiol. 25:399405. 14. Lussier, G. 1962. Vibrionic dysentery of swine in Ontario. I. Clinical aspects of pathology. Can. Vet. J. 3:237-288. 15. Whipp, S. C., D. L. Harris, J. M. Kinyon, K. G. Songer, and R. D. Glock. 1978. Enteropathogenicity testing of Treponema hyodysenteriae in ligated colonic loops of swine. Am. J. Vet. Res. 39:1293-1296. 16. Whiting, R. A., L. P. Doyle, and R. S. Spray. 1921. Swine dysentery. Purdue Univ. Agric. Exp. Stn. Res. Bull. 257:3-15.
