The Etiology of Swine Dysentery

Transcription

1 Vet. Pathol. 12: (1975) The Etiology of Swine Dysentery 111. The Role of Selected Gram-Negative Obligate Anaerobes R. C. MEYER, J. SIMON and C. S. BYERLY Department of Veterinary Pathology and Hygiene, University of Illinois at Urbana-Champaign, Urbana, Ill. Abstract. A disease resembling swine dysentery occurred in gnotobiotic piglets previously colonized with Escherichia coli and given a mixture of five enteric anaerobes and spirochete B-78, orally. No disease occurred in germ-free piglets exposed to four Gramnegative obligate anaerobes or to the spirochete; however, if the four respective anaerobes were introduced into gnotobiotic pigs previously colonized with the spirochete, or when the spirochete was introduced into gnotobiotic pigs colonized with the anaerobes, a disease occurred that simulated swine dysentery. Swine dysentery is probably caused by the specific interaction of one or more enteric Gram-negative obligate anaerobes and a spirochete. Although a spirochete has been implicated as the cause of swine dysentery [I, 4, 5, 171, previous studies [ll, 121 with gnotobiotic swine indicated that more was involved than an intestinal spirochete. The evidence strongly suggests that the production of the clinical disease requires the combined if not synergistic action of two or more agents. A number of microbial agents that constitute part of the normal flora of an animal may function as conditional or opportunistic pathogens. There is little information available on the normal, Gram-negative anaerobic intestinal flora of swine, however, particularly as it relates to the fusobacteria and bacteroides and their actual or potential role in diseases of swine. There are similarities between swine dysentery and other fusospirochetal, necrotizing infections of the mucous membrane. Because of our previous studies [l 1, 121 and knowledge of mixed anaerobic infections of the mucous membranes in man [8], it appeared logical to examine a number of anaerobes (bacteroides and fusoforms) of swine origin to determine their possible role in the cause of swine dysentery. This report discusses the production of a swine-dysentery-like disease in germ-free swine inoculated with a known mixture of selected microbes.

2 MEYER/SIMON/BYERLY 47 Materials and Methods Equipment, methods of procurement, rearing, monitoring, and general procedures for germ-free swine were as previously described [9,10]. The microbial agents consisted of the spirochete B-78, Escbericbiu coli, a clostridium, and four Gram-negative obligate anaerobes. All seven agents were of swine origin. The four Gram-negative anaerobes, however, have not been fully characterized and currently have been designated tentatively either as species of bacteroides or fusobacteria. The details of their isolation and characterization are in another report. For convenience in this study these agents have been designated as agent 1 (bacteroides), agent 2 (fusoform), agent 3 (fusoform), and agent 4 (tentatively, Bacteroides meluninogenicus). The E. coli and clostridial sp. were the same as used in our previous study [12]. Cultivation of the spirochete, E. coli and Clostridiu and preparation of inoculum were reported previously [I 1, 121. The four Gram-negative anaerobes were cultured on a reduced (infusion-trypticase) medium enriched with rumen fluid, laked defibrinated bovine blood and vitamin K. Incubation was anaerobically at 36 C using anaerobic jars (BBL Gas Paks, Cockeysville, Md.). The inoculum of the anaerobes consisted of 72- to 96-hour broth cultures of each, administered orally in 2-ml volumes by mixing in 150 ml of milk and fed to the piglets. Two separate trials were conducted using pigs from two different litters. In trial I four 5-week-old piglets colonized with E. coli 2 weeks earlier as part of an unrelated study were inoculated with the spirochete, the clostridium, and four Gramnegative anaerobes. To facilitate colonization an identical inoculum was administered a second time 3 days later. One similar gnotobiotic piglet colonized only with E. coli served as the negative control. The piglets were observed three times a day and examined daily for signs of diarrhea or changes in the character of their stools. Two pigs were killed and necropsied 8 days after the initial oral inoculation. The two remaining inoculated pigs plus control were killed on the 11 th day. In trial I1 the experiment was conducted in two phases. Six 28-day-old germ-free piglets were bled and divided into three groups of two each (table 1). In the first phase two piglets (group B) were inoculated with the spirochete. The remaining four (groups A and C) with the four Gram-negative anaerobes. As in the previous trial the respective microbial agents were administered a second time 3 days later to facilitate colonization. One germ-free piglet was left as an uninoculated control. All piglets were observed for the next 9 days for any signs of enteric disease. In the second phase of the experiment the two piglets colonized with the spirochete were now exposed to the four anaerobes, and two of the pigs colonized with theanaerobes were inoculated with the spirochete. The six piglets, four with the spirochete-anaerobe combination and two with the anaerobes only, were observed as before for changes in the character of the stools and signs of swine dysentery. One representative pig from each group (A, B, and C) was killed 14 days after exposure to the combination of the anaerobes and spirochete. The remaining pigs were killed 17 days after exposure to the mixture (table I). Pigs were bled after an overdose of sodium thiopental. All necropsies were done aseptically. Tissues for histopathologic examination were fixed in neutral 10% formalin and consisted of brain, heart, lung, liver, spleen, kidney, stomach, segments of the small intestine, cecum, and colon. Twenty-four hours after

3 48 MEYER/SIMON/BYERLY Fig. I. Section of colon from pig killed 8 days after inoculation with test organisms. Trial 1. Large amount of mucus readily removed from mucosal surface (arrow). fixation the tissues were trimmed, processed on an autotechnicon model 2A, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin. Additional representative areas of the intestinal tract were also stained using Warthin-Starry's method. Results In trial I, signs of enteric disease became apparent 5 days after inoculation with the combined agents. Intestinal signs became progressively more pronounced over the next 3 days so that by the 8th day the stools were mucopurulent. Considerable mucus was seen in the colon of a pig killed 8 days after inoculation (fig. 1). By the 1 lth day after inoculation, the exudate became much more purulent with formation of a distinct pseudomembrane composed of fibrin, inflammatory cells, bacteria, and necrotic mucosal cells (fig. 2). In trial I1 piglets infected with either the spirochete or Gram-negative anaerobes alone remained free of swine dysentery during the initial 9-day observation period (phase I). Upon introduction of the anaerobes into pigs colonized with the spirochete or the reverse situation, signs of disease became evident on the 4th and 5th days. As in trial I the first sign in afflicted pigs was considerable mucus in their stools. The two piglets inoculated with the anaerobes only, however, remained normal throughout the study. In pigs killed 14 and 17 days after complementation (anaerobes plus spirochete), both gross and histologic changes consistent with early acute swine dysentery as seen in the field were present (fig. 3-5).

4 Etiology of Swine Dysentery 49 Fig. 2. Section of colon from gnotobiotic pig in trial I, 11 days after inoculation with test organisms. Extensive catarrhal exudate mixed with inflammatory cells and debris. Hematologic studies of pigs bled before and after exposure to the various agents indicated that the only pigs that showed a significant leukocytosis and left shift were those exposed to the complete complex of anaerobes and spirochete. Pigs exposed to only the anaerobes or spirochete alone showed no change indicative of an infectious process. There was extensive catarrhal exudation, mixed with necrotic epithelial cells, bacteria, fibrin, debris, and focal necrosis of the epithelial lining of the more superficial glands. These changes, although similar to those in early cases of swine dysentery, were not as severe as those seen in natural outbreaks of the disease and lacked the degree of coagulation necrosis and hemorrhage often viewed in such outbreaks. The respective microbial agents used in the study were re-isolated from afflicted piglets and readily seen in stained smears of colonic scrapings. Discussion This experiment provided additional evidence that swine dysentery is probably a mixed infection and that the production of clinical disease depends

5 50 MEYER/SIMON/BYERLY c Fig. 3. Section of colon from pig used in trial I1 showing necrosis of the epithelium; 14 days after inoculation with anaerobes and spirochete. on the interaction of one or more obligate anaerobes in combination with an intestinal spirochete. Whether other microbial agents besides the four anaerobes used in this study can fulfill the requirements essential for disease production or if in fact all four of the anaerobes are really needed is open to question. The approach used in this study does provide a means to identify more precisely the various agents involved in this disease. In preliminary studies [unpublished data] mixtures comprising cultures of as many as 14 different agents, including a large number of Gram-positive agents, were evaluated as to their disease-producing capacity. By removing various agents from such mixtures we have been able to narrow the number of agents involved to the four Gram-negative, obligate anaerobes and the spirochete used in this study. The elimination of the Gram-positive agents was a little surprising in that many synergistic mixed infections have been reported to include them [8, 13, 15, 161. The value of the germ-free pig in studies of this type has again been amply demonstrated ; it is doubtful that any other procedure could have demonstrated the interrelationship between the anaerobes and the spirochete. Indeed, were it not for the failure of disease to develop after the colonization of germ-

6 Etiology of Swine Dysentery 51 Fig. 4. Section of colon showing dilated, cystic gland containing hyalinized debris, and catarrhal exudate projecting into the lumen. Trial 11, 17 days after inoculation. free piglets by the spirochete [I 11, the apparent requirement for select anaerobes in this disease might have been overlooked. One of the agents (agent 3) used in this study is thought to be B. meluninogenicus, a pathogen of man now recognized to be a principal agent in mixed infections of mucous membrane [2,6,8, 161. Since this strain was originally isolated from colonic scrapings obtained in an experimental case of swine dysentery [12], it may very well prove to be essential for the initiation of clinical disease [7,8]. The establishment of B. meluninogenicus in the germfree pig occurred with relative ease, in contrast to the reported failure of this agent to become established in germ-free mice [8]. The successful establishment in the pig, however, may be a reflection of the fact that this strain was of swine origin and as such might have been better adapted to the conditions provided by this species. It has been thought that spirochetes, when present in mixed infections, are of special significance and confer a unique character to such infections, as the term fusospirochetal implies. The role of various spirochetes in such infections, however, has been questioned. The arguments against the spiro-

7 52 MEYER/SIMON/BYERLY Fig. 5. Higher magnification of area shown in figure 4. Many polymorphonuclear leukocytes between the glands and mixed with debris on the mucosal surface. chete being a primary pathogen in acute ulcerative gingivostomatitis in man are convincing [3, 81. In this regard, the spirochete associated with swine dysentery also does not appear to be a true pathogen in the classical sense [l 13. It should be emphasized that in this particular study the spirochete was employed in its normal host. In much of the work done on fusospirochetal infections of man, the experimental host was one of the standard laboratory animals (guinea pig, hamster, rabbit). Does failure to demonstrate a pathogenic potential in an experimental host necessarily mean the agent has no function in the disease as it occurs in the natural host? This question can not be answered here. The spirochete associated with swine dysentery, however, does appear to play an active, if not synergistic role in the etiology of the disease as shown by the fact that disease did not occur in its absence (table I). What this role may be is unknown. Progress has been made in clarifying the major etiologic agents in what is considered to be swine dysentery. In the field, however, swine dysentery does not always present a uniform clinical picture. Because we have greatly reduced and controlled the kinds and numbers of agents in these experi-

8 Etiology of Swine Dysentery 53 Table I. Production of disease in germ-free piglets; trial 11, role of the anaerobes-spirochete mixture Germ-free pigs, Phase I Phase I1 group agent($ clinical agent(s) clinical inoculated disease inoculated disease A anaerobes neg. spirochete pos.8 B spirochete neg. anaerobes pos.' C anaerobes neg. - neg.' Control - neg. - neg. Observed for 9 days after inoculation. Observed 14 and 17 days after second inoculation to complete the complex. Total elapsed time (phase I and 11) was 26 days. ments, we recognize that the disease produced here in a defined system cannot be truly representative of what may occur under field conditions. Various microbial agents may participate in what is called swine dysentery, and various opportunists could modify both the course and severity of the condition [14]. The chronic form might well result from the activities of opportunists not necessarily involved in the initiation of the early or acute phase of the disease. Substantial work is needed to delineate all the potential interactions, particularly when contemplating the complexity of the intestinal flora. The recognition of the role of the obligate anaerobes should aid in the study of the biochemical nature of the synergistic relationship of the anaerobes and spirochete. Ackno w Iedgements Supported by funds from the swine disease research program, Illinois Department of Agriculture, and by a grant from the National Pork Producers Council. References 1 AKKERMANS, J.P. W.M. and POMPER, W.: Etiology and diagnosis of swine dysentery (Doyle). Tijdxhr. Diergeneesk. 98: (1973). 2 BIBERSTEIN, E. L.; KNIGHT, H.D., and ENGLAND, K.: Bacteroides melaninogenicus in diseases of domestic animals. J. Am. vet. med. Ass. 153: (1968).

9 54 M EY ER/SIMON/BY ERLY 3 FITZGERALD, R. J.; HAMP, E.G., and NEWTON, W.L.: Infectivity of oral spirochete in cortisone treated and germfree guinea pigs. J. dent. Res. 37: (1958). 4 HAMDY, A.H. and GLENN, M.W.: Transmission of swine dysentery with Treponemu hyodysenteriue and Vibrio coli. Am. J. vet. Res. 35: (1974). 5 HARRIS, D.L.; CLOCK, R. D.; CHISTENSEN, C. R., and KINYON, J. M. : Swine dysentery. I. Inoculation of pigs with Treponemu hyodysenferiue (new species) and reproduction of the disease. Vet. Med./Small Anim. Clin. 67: (1972). 6 HEINRICH, S. und PULVERER, G. : Uber das physiologische Vorkommen des Bucferoides meluninogenicus bei Mensch und Tier. Z. Hyg. InfektKrankh. 146: (1960). 7 KAUFMAN, E. J.; MASHIMO, P. A.; HAUSMANN, E.; HANKS, C.T., and ELLISON, S. A.: Fusobacterial infection enhancement by cell free extracts of Bucteroides meluninogenicus possessing collogenolytic activity. Archs oral Biol. 17: (1972). 8 MACDONALD, J.B.; SOCRANSKY, S.S., and GIBBONS, R. J.: Aspects of the pathogenesis of mixed anaerobic infections of mucous membrane. J. dent. Res. 42: (1963). 9 MEYER, R.C.; BOHL, E.H.; HENTHORNE, R.D.; THARP, V.L., and BALDWIN, D.E.: The procurement and rearing of gnotobiotic swine. Lab. Anim. Care 13: (1963). 10 MEYER, R.C.; BOHL, E.H., and KOHLER, E.M.: Procurement and maintenance of germfree swine for microbiological investigations. Appl. Microbiol. 12: (1964). 11 MEYER, R.C.; SIMON, J., and BYERLY, C.S.: The etiology of swine dysentery. I. Oral inoculation of germ-free swine with Treponemu hyodysenteriue and Vibrio coli. Vet. Pathol. 11: (1974). 12 MEYER, R.C.; SIMON, J., and BYERLY, C. S.: The etiology of swine dysentery. 11. Effect of a known microbial flora, weaning and diet on disease production in gnotobiotic and conventional swine. Vet. Pathol. 11: (1974). 13 MOORE, W.E.C.; CATO, E. P., and HOLDEMAN, L.V.: Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infections. J. infect. Dis. 119: (1969). 14 RAYNAUD, J. P.; RENAULT, L.; MARIE, C. et VAISSAIRE, J. : Reproduction exptrimentale de I enttrite htmorragique (dysenterie) du porc en France. Revue Mtd. vtt. 123: (1972). 15 ROBERTS, D.S.: Synergic mechanisms in certain mixed infections. J. infect. Dis. 120: (I 969). 16 SOCRANSKY, S.S. and GIBBONS, R. J.: Required role of Bucferoides nieluninogenicus in mixed anaerobic infections. J. infect. Dis. 115: (1965). 17 TAYLOR, D. J. and ALEXANDER, T. J.L.: The production of dysentery in swine by feeding cultures containing a spirochete. Br. vet. J. 129: (1971). Dr. R. C. MEYER, Department of Veterinary Pathology and Hygiene, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbunu, IL (USA)