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Mitigation of Experimental Inflammatory Bowel Disease in Guinea Pigs by Selective Elimination of the Aerobic Gram-Negative Intestinal Microflora
67: 460- 472, 1974 by The Willi a ms & Wilkins Co.
Vol. 67 . No. :3
GASTROENTEROLOGY
Copyri ght ©
197~
Printed in U.S.A.
MITIGATION OF EXPERIMENTAL INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS BY SELECTIVE ELIMINATION OF THE AEROBIC GRAM-NEGATIVE INTESTINAL MICROFLORA D. VAN DER WAAIJ, M.D., PH .D ., B. J. COHEN, D.V.M. , PH.D. , AND M . R. ANVER D.V .M ., PH.D. Unit for Laboratory Animal Medicin e, University of A1ichiga n , Ann Arbor, Michigan
Experimentally induced ulcerative disease of the large intestine in guinea pigs was significantly mitigated by selective elimination of the aerobic gram-negative intestinal microflora. Lesions were induced with 2 or 5% degraded carrageenan administered in the drinking water for 30 to 44 days. Craterous cecal or colonic ulcers, crypt abscesses, mucosal distorti on, mesenteric lymphadenopathy, marked cecal or colonic lymphoid hyperplasia , and other lesions were seen in conventional guinea pigs treated with degraded carrageenan . Other guinea pigs that were treated with 2% degraded carrageenan, but freed of Enterobacteriaceae species by the administration of trimethoprim with sulfamethoxazole, and subsequently maintained in isolators, exhibited virtually no lesions. Enterobacteriaceae-free animals that were treated with 5% degraded carrageenan had significantly fewer lesions than were observed in the conventional degraded carrageenan-treated animals. Biotyping of bacterial isolates indicated that the family of Enterobacteriaceae species generally, and not a single biotype, was associated with the lesions. The bacteria presumably stimulated an immunological response after penetration of the intestinal mucosal barrier ; thus , inflammatory bowel disease in guinea pigs, induced with degraded carrageenan , may provide a useful model for investigating immunological aspects of human inflammatory bowel disease. rhesus monkeys. t- s Initially, Watt and Marcus 1 induced ul cerations of the colon in guinea pigs that were fed 1% aqueous extracts of the dried red seaweed, Euchema spinosum, for 5 months. Subsequently, similar ulcerative lesions were produced within 30 days with 1 to 5% aqueous solu-
In recent years, ulcerative disease of the large intestine has been induced experimentally in several animal species, including guinea pigs, rabbits, rats, mice, and Received December 17, 1973. Accepted February 26, 1974 . Address requests for reprints to: Dr. Bennett J. Cohen, Unit for Laboratory Animal M edicine, 010 Animal Research Facility, University of Michi gan , Ann Arbor, Michiga n 48104. This investigation was s upported in p art by grants from the Anim al Resources Branch, Di vision of Research Resources, National Institutes of Health (RR 00200), from the University of Michigan Cancer Research Committee, and from National Science Foundation Institutional Grant (GU-3470). Dr. van der Waa ij's present address is: Radiobiological Institute, TNO, 151 Lange Kleiweg, Rij swijk (ZH), The Netherlands . The degraded carrageenan used in this study was
generously provided by Dr. G. B. Shirlaw and Dr. J. Thomas, Laboratoires Glaxo, S. A., Paris, Fra nce. The technical assistance of J ong Soo Park is gratefully ack nowled ged. The guinea pigs were housed in facili ties that are fully accredited by the American Assoc iation for Accreditation of Laboratory, Animal Care (AAA· LAC), and were ma inta ined in accordance wi t h stan· d ards in the Guide for the Care and Use of Lab oratory Animals. Departm en t of Health, Education a nd Wei· fare publication (National Institutes of Hea lth ) 73-23, Revised 1972.
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
tions of carrageenan, derived from the same seaweed but degraded by mild acid hydrolysis. 2 • 3 The clinical and pathological features of the experimentally induced disease resembled those of human ulcerative colitis in many respects, thus providing a simple model for investigating ulcerative disease of the colon. Several investigators have recently indicated the involvement of Escherichia coli (0119: B14) in the pathogenesis of human ulcerative colitis. Watson et al. 7 demonstrated that circulating lymphocytes, but not sera, from ulcerative colitis patients are cytotoxic in vitro for human adult autologous and allogeneic colonic epithelial cells. Shorter et al. 8 induced cytotoxicity for allogeneic colonic epithelial cells by preliminary incubation of lymphocytes derived from peripheral blood of normal healthy adults, with a lipopolysaccharide extract from E. coli (0119:B14). They suggested that the colonic ulcerations, with the resulting massive exposure of the individual to microorganisms from the lumen, may be an important factor in the induction of the cytotoxic property of the lymphocytes. Furthermore, these investigators speculated that this cytotoxicity may be introduced through shared antigenicity between bacteria and tissue constituents which may not be confined to E. coli (0119:B14). Thus, E. coli in particular, and Enterobacteriaceae species and other potentially pathogenic microorganisms in general, may play a significant role in the pathogenesis of ulcerative colitis. Ulcerative colitis patients often have a history of a bacterial infection of the large bowel. 8 • 9 The infection is always associated with bacterial invasion of the intestinal mucosa and an increase in the concentration of the causative bacterium in the feces. This suggests the possibility that a decrease in the colonization resistance of the digestive tract, which is also associated with an increase in concentration of potentially pathogenic intestinal bacteria, 10 is a factor in the pathogenesis of the lesions of ulcerative colitis. However, bacterial invasion of the intestinal mucosa may occur under a variety of conditions. For example, a high fecal concentration of a specific
461
bacterium has been demonstrated in mice, with subsequent passage of the bacterium through the intestinal mucosa into the mesenteric lymph nodes and spleen, after massive oral contamination with the bacterium. 11 The principal objective of this investigation was to determine the possible involvement of aerobic gram-negative intestinal bacteria in the pathogenesis of large intestinal ulcerations induced in guinea pigs with degraded carrageenan. We also attempted to link the occurrence of lesions with one or more specific bacteria, such as E. coli. Finally, we sought .to determine whether the administration · of degraded carrageenan to guinea pigs affects colonization resistance and the concentration of the aerobic gram-negative int~stinal microflora. Experimental Procedures Animals
Thirty-five male, English short-hair, albino guinea pigs were used. The animals were 3 to 7 months old and weighed 400 to 820 g. Each guinea pig was weighed twice weekly throughout the study . Housing and Care
Twenty-nine of the guinea pigs were housed under conventional conditions in a separate room in individual stainless steel cages equipped with grid floors. Six guinea pigs which were freed of Enterobacteriaceae, but which were not otherwise "germ free ," were maintained in identical cages or in clear plastic "shoebox" cages inside flexible plastic isolators. 12 The isolators were sterilized with 2% peracetic acid before the guinea pigs were introduced into them. The guinea pigs were fed a commercially available pelleted ration ad libitum. The diet was autoclaved at 121 C for 20 min to prevent any possibility of bacterial contamination. The drinking water was provided daily in bottles equipped with stainless steel sipper tubes . Ascorbic acid was added (0 .2 mg per ml) to compensate for the loss of vitamin C in the autoclaved diet. The drinking water also was used as the vehicle both for degraded carrageenan and the agent used to eliminate the Enterobacteriaceae from the intestinal flora; accordingly, sucrose (0 .05 g per ml) also was added to increase palatability.
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VANDER WAAIJ ETAL.
Degraded carrageenan (Laboratoires Glaxo, Paris, France) was administered to conventional and Enterobacteriaceae-free guinea pigs in a 2 or 5% solution in the drinking water for 30 to 44 days (table 1) . The solutions were prepared fresh daily for the conventional guinea pigs. For the Enterobacteriaceae-free guinea pigs , stock solutions were prepared twice weekly under strict aseptic conditions using sterilized water. The guinea pigs were supplied daily from the stock solutions which were stored in the isolators. Trimethoprim with sulfamethoxazole, administered as Bactrim (Hoffmann-La Roche, Nutley , N.J.) was added to the stock solutions (0.25 mg per ml) to prevent multiplication of Enterobacteriaceae in the event of accidental contamination inside the isolators.
Microbiological Methods Elimination of Enterobacteriaceae species from the guinea pigs' intestinal microflora. Fresh feces from a group of 15 guinea pigs were cultured initially to determine the presence and concentration of aerobic gram-negative bacteria. Ten of these guinea pigs were shown to be free of Pseudomonas aeruginosa, which is resistant or has a low sensitivity to trimethoprim with sulfamethoxazole. However, this agent is highly effective against the Enterobacteriaceae. The Enterobacteriaceae flora of the 10 Pseudomonas-free guinea pigs was investigated for its sensitivity to trimethoprim with sulfamethoxazole by plating 6-hr broth cultures of fresh feces on Endo agar (Difco, Detroit, Mich.). Sensitivity discs (25, 50, 100 llg per disc) were placed on the plates before they were incubated overnight at 37 C. The Enterobacteriaceae microflora of the 10 TABLE
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guinea pigs was shown to be sensitive to trimethoprim with sulfamethoxazole, as indicated by an inhibition zone of more than 10 mm around all of the corresponding sensitivity discs for each animal. These guinea pigs then were transferred into a separate room where special precautions were taken to prevent contact contamination. Trimethoprim with sulfamethoxazole was administered in the drinking water (2 mg per ml) for 4 days, at which time 7 of the 10 guinea pigs were free of Enterobacteriaceae, as demonstrated by negative fecal cultures. These animals were then transferred into three isolators in groups of 2 or 3, and administration of trimethoprim with sulfamethoxazole was con tinued for an additional 10 days. Fresh feces were cultured daily to determine whether the guinea pigs remained free of aerobic gram-negative bacteria. Bacteriological culturing. Feces. Fresh feces were collected daily. Samples weighing approximately 100 mg were suspended in 0.5 ml of brain heart infusion broth (Difco). Subsequent serial dilutions were made with 0.05-ml diluting loops (Flow Laboratories , Rockville, Md.) in a plastic tray containing rows of eight cups, each filled with 0.5 ml of brain heart infusion broth. The diluting loops were heat-sterilized between dilution steps. The trays were placed in a plastic container and covered with aluminum foi l to prevent dehydration. After incubation overnight at 37 C, the various dilutions were subinocu lated on Endo agar plates which were incubated overnight at 37 C. The concentration of Enterobacteriaceae and Pseudomonas in the feces was interpreted from the growth observed on these plates. Water . Water from the drinking bottles of the conventionally housed guinea pigs was sampled
1. Experimental groups and treatments
No. examined Condition of guinea pigs
Degraded carrageenan treatm ent
Total no. of animals
Bacteriologically Daily feca l Enterobacteriaceae
Histologically Biotyping
concentration
Conventional Conventional Free ofEnterobacteriaceae and Pseudomonas Total
Controls; no degraded carrageenan administration 2% for 30- 44 days 5% for 30 days 2% for 37 days 5% for 30 days
9
5
5
9
13 7 4 2
10 7
10 7
13 7 4 2
35
28
22
35
4 2
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
before and after rinsing and refilling, and was cultured quantitatively in the same way as the fecal suspensions. Subinoculations on Endo agar were performed to determine the original concentration of gram-negative bacteria in the water samples, also in the same way as for the fecal suspensions. Biotyping. Once weekly a sample of fresh feces from each guinea pig was streaked directly on Endo agar and cultured in brain heart infusion broth. After 6 and 24 hr of incubation at 37 C, the broth cultures were subinoculated on Endo agar. The culture plates were examined after incubation overnight at 37 C. As many different looking colonies as possible from each plate were selected for subinoculation, with a minimum of 12 per sample. The colonies were subinoculated on other Endo agar plates to obtain pure cultures. The cultures thus obtained were subinoculated on Kligler agar slants (Difco) to further assure purity. Finally, the pure cultures were suspended in sterile saline, and these suspensions were used to inoculate sets of a commercially available series of biochemical and cultural tests (API 20 Enteric, Analytab Inc ., New York, N. Y.). This test series is useful in differentiating Enterobacteriaceae and other aerobic gram-negative bacteria. After inoculation, the test strips were incubated for 24 hr at 37 C, and the various reactions were interpreted in accordance with a key which is provided with the strips. The principles of this technique were identical to a previously described technique for biotyping Enterobacteriaceae, 13 although not all of the biochemical tests were identical. The results of the biochemical tests were coded by means of a binomial system which yielded an identification number for each of the various biotypes. 13 Arbitrary digital numbers were assigned in sequence at the time the 12 or more colonies were selected for biotyping. A graph was constructed to aid in determining whether the selection of 12 or more than 12 colonies was needed to assure an optimal inventory of the biotypes in each test sample (fig. 1). The abscissa of the graph described the number of morphologically different colonies typed; the ordinate identified the number of different biotypes in each group of four colonies. The 12 colonies originally selected for typing were divided arbitrarily into three sequential groups of four. The number of different biotypes in the first group of four was plotted , as illustrated in figure 1. The number of biotypes in the second group of four colonies that differed from those found in the first group of colonies, and from each other, also was plotted. This process was
q·· . . ~
f:::r--6 Sample of 5/14/73 o ....
o
Sample o f 6/4/73
~ 8
12
16
NUMBER OF MORPH OLOGIC ALLY DIF FERENT COLONIES TYPE D
FIG. 1. The number of colonies whose biotypes must be determined to assure an optimal inventory of each test sample. The curves derived from two tests on 1 guinea pig are shown. The results indicate that the biotyping of 12 colonies provided a complete inventory.
continued until the different biotypes in all three groups were similarly plotted. A "best fitting" curve was then drawn through the points. The intersection of this curve with the abscissa indicated the minimum number of colonies on which biotyping was needed to identify virtually all of the biotypes in the test sample . Preliminary tests, as illustrated in figure 1, indicated that the selection of 12 colonies usually was adequate to provide this inventory. Thus, 12 colonies were routinely biotyped; if, however, the curve did not reach the abscissa at the third group of colonies, additional colonies were selecte d for biotyping from the original Endo agar plates that had been stored at 4 C.
Necropsy and Histological Methods At the end of the experimental period each guinea pig was killed with C0 2 gas and necropsied immediately. The thoracic and abdominal viscera were examined grossly. Sections of the various organs were fixed in 10% buffered neutral formalin. The gastrointestinal tract was removed in its entirety. The cecum and colon were emptied of feces and inspected for gross evidence of ulcerations or other lesions. Stomach, duodenum, jejunum, ileum, cecum, proximal colon, midcolon, and distal colon also were fixed in formalin for subsequent histological examination. Fixed tissues were embedded in paraffin, sectioned at 5 !J. , stained with hematoxylin and eosin, and examined routinely by light microscopy. For histochemical identification of pigment, representative sections of cecum, colon, mesenteric lymph nodes, and spleen were also stained with periodic acid-
464
VANDER WAAIJ ET AL.
Schiff, Sudan Black B for lipid , and Perls' Prussian blue for iron .
Results Clinical Course The conventional guinea pigs drank nearly twice as much of the 2% degraded carrageenan solution t han they did the 5% solut ion (table 2). As a result, the actual daily intake of degraded carrageenan was similar for both groups (tab le 3) . The intakes recorded for the Enterobacteriaceae-free animals were identical. A few guinea pigs refused the 5% solution and became severely dehydrated. These animals were removed from the experiment and are not included in the results. The clinical course of the conventional guinea pigs was similar to that described by Watt and Marcus. 3 Animals receivi ng 2 or 5% degraded carrageenan invariably exhibited weight loss or a sharpiy flattened growth curve starting within several days to 2 weeks after administration of degraded carrageenan was begun. The weight loss was greater in t he animals given 5% degraded carrageenan. All of the guinea pigs which received 5% degraded carrageenan, TABLE
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and many but not a ll of those that received the 2% solution, exhibited soft feces during the period of degraded carrageenan administration. Blood was grossly visible in the feces of 3 of the 7 animals which received the 5% solution, but in only 2 of 13 animals which received 2% degraded carrageenan. All 4 of the Enterobacteriaceae-free guinea pigs which received 2% degraded carrageenan gained weight throughout the period of administration and produced normally firm fecal pellets. The 2 Enteroba cteriacea-free guinea pigs which received 5 9C degraded carrageenan lost weight during the period of administration. Soft feces were noted sporadically, but during most of the period of degraded carrageenan administration these 2 animals produced firm but smaller than normal fecal pellets . Necropsy and Histologica l Findings Consistently found at necropsy in the conventional guinea pigs which received 2 or 5% degraded carrageenan were innumerable white foci of varying sizes that were visible grossly through the serosa of the cecum and colon (tab le 4, fig . 2B). The histological picture was that of nor-
2. Concentration of aerobic gram-negative bacteria in the drinking water of conventional guinea pigs Mean log concentration of gra m- nega ti ve
% Degraded
No . of
carrageenan
in the water
a nim als
0 2 5
5 10 7
bacteria/m l" Water intake" Just before refilling water bottl es
Directl v after refilling wa.ter bot tles
5.7 (4- 7) 6.3 (4-7) 6.0 (4-7)
:<; 1 :<; 1 :<; 1
ml/day
a
85.0 (68- 100) 74.0 (59-97) 38.5 (30- 44)
Numbers in parentheses a re range.
TABLE
3. Daily dose of degraded carrageenan and colonization properties of aerobic gram-negative bacteria isolated from fec es of conventional guinea pigs Mean and range of:
Degraded carrageenan in water
No. of animals
Body wt at start of experiment
carragee n an
Log conce ntration of gram-negative
No . of hioty pes isola ted once
No. identical biotypes isolated
1.4 (0-4) 2.7 (1-5) 0.1 (0-1)
1.0 (0. 2) 4.1 (3.9- 5.4) 4.0 (3- 5)
bacteria/g of feces
repeated!~·
g/kg body wt
%
0 2 5
Da ily dose of degrad ed
5 10 7
780 (71 5-870) 550 (500-610) 820 (740-920)
2.5 (2.1 -3.3 ) 2.4 (1.7- 2.8)
1.7 (1.5-1.9) 4.1 (3.9-5.4) 4.5 (4.0-4.9)
September 1974 TABLE
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
4. L esions in conventional and Ent erobacteriaceae-free guin ea pigs treated with degraded carrageenan Enterobacteriaceae·free
Conventional Treated with degraded ca rrageenan
Lesion
Treated with d egraded ca rrageenan
Unt reated
In creased cecal or colon ic submu cosal lymphoid tissue Enlarge ment of cecal or co lonic ly mph nodes Crypt abscesses Cec um Distal co lon Ulcers Cecum Distal colon Polymorp honuclear leukocytes in lam in a propria Cecum Distal colon Mucosal di stortion Cec um Dista l colon Mucosal a nd submucosal gra nulation tissue Cec um Distal co lo n Mesenteri c fibros is
2%30-44 days
5% 30 days
2%37 days
5% 30 days
1/ 9"
11/ 13
7/7
1/ 4
1/2
0/ 9
3/13
6/7
0/ 4
2/2"
0/ 9 0/7
1/ 13 1/13
5/7 4/7
0/ 4 0/4
1/2 0/ 2
0/9 0/7
3/13 5/13
2/7 4/7
0/4 0/4
1/2 1/2
2/9 2/7
8/13 5/ 13
4/ 7 5/7
0/4 0/ 4
2/2 0/ 2
0/ 9 0/7
2/13 5/13
4/7 4/7
0/ 4 0/ 4
1/2 1/2
0/ 9 0/7 0/ 9
4/ 13 5/13 1/ 13
4/7 4/7 4/ 7
0/ 4 0/4 0/4
1/2 1/2 0/2
"No. with les ions per total of no. exam in ed. • Slight e nl a rge ment.
mally active submucosal lymphoid tissue; but the number of such foci was far greater than in conventional untreated guinea pigs (fig. 2A). Two of the degraded carrageenan-treated Enterobacteriaceae-free guinea pigs also exhibited a moderate increase in cecal or colonic lymphoid patches, and other lesions (table 4) also were present. However, this group of guinea pigs generally was more comparable to untreated controls than to degraded carrageenan-treated conventional animals. Typical findings (table 4) in the degraded carrageenan-treated conventional guinea pigs were: enlarged cecal or colonic lymph nodes, cecal or colonic mucosal crypt abscesses and mucosal distortion (fig. 3), gross and microscopic craterous ulcers (figs. 4 and 5), polymorphonuclear leukocytes in the lamina propria, mucosal and submucosal granulation tissue, and fibrosis of the mesentery (fig. 6). The lesions were
more extensive and occurred more frequently in the guinea pigs treated with 5% degraded carrageenan than in those treated with the 2% solution. The ulcers in these conventional guinea pigs also were more severe and widespread in the distal colon than in the cecum . Ulcers were not seen in the proximal colon. In general, the ulcerative lesions closely resembled those described by Watt and Marcus . 3 In addition, dense deposits of iron-positive pigment, interpreted as hemosiderin, were demonstrated consistently in the submucosa of the proximal colon and cecum, but only infrequently in the distal colon, both of conventional and Enterobacteriaceae-free guinea pigs which had received degraded carrageenan . The pigment was similarly present, but to a much lesser extent, in conventional untreated animals. In 4 of the 6 degraded carrageenan-treated Enterobacteriaceae-free guinea pigs, focal
466
VANDER WAAIJ ET AL.
hemorrhages were present m the lamina propria or submucosa. Microbiological Results The feces of the guinea pigs in the isolators remained free of Enterobacteriaceae species and of Pseudomonas aeruginosa throughout the experiment.
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The only aerobic microorganisms isolated from these animals were a few unidentified bacillus species and a yeast, Candida albicans. Candida was present in a concentration of 10 4 to 10 8 per g of feces in the two 5% degraded carrageenan-treated Enterobacteriaceae-free animals . Two of the 2% degraded carrageenan-treated En-
FIG. 2. A, cecum of a n unt reated, control guinea pig with a few , scattered lymphoid foci ( x 1.5). B, multiple white ly mphoid foci see n through the cecal serosa in a 5% degraded carrageenan-treated gu inea pig. Compare with A ( x 0.5) .
-111111 .11 -llllllllll t II
II JIll I II1111111 1' Ill t-lllll !11 111111 !II II
FIG. 4. Distal colon mucosa of 5% degraded carrageenan-treated animal with multiple ulcers ( x 3).
467
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VANDER WAAIJ ET AL.
terobacteriaceae-free animals were negative for Candida, while 2 had fecal concentrations up to 10 4 per g. In the conventional guinea pigs, yeasts were only occasionally found, and only in concentrations less than 10 2 per g. In the conventional guinea pigs, the concentration of Enterobacteriaceae species in the feces invariably increased within
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the first 3 days, and remained high throughout the degraded carrageenan treatment period (table 3). These animals were colonized with a relatively small num· her of different biotypes (fig. 7). Six different klebsiella biotypes were isolated, one of which was found in all 17 animals, while another occurred in 7 animals; the remain· ing four biotypes occurred less frequently.
FIG. 5. Chronic ulcer in distal colon of 5% degraded carrageenan·treated animal. Calcification (large arrow) and fibrosis at base of ulcer with masses of overlying polymorphonuclear leukocytes . Muscularis mucosae (small arrow) intact; submucosa contains many foamy macrophages and polymorphonuclear leukocytes (hematoxylin and eosin. x 430).
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
469
FIG. 6. Fibrosis and foamy macrophages in the mesentery of an ulcerated distal colon from a guinea pig treated with 5% degraded carrageenan (hematoxylin and eosin, x 100) .
E. coli was represented by five different biotypes, while only two different biotypes of Pseudomonas aeruginosa were isolated in repeated weekly samples from 2 of the 17 guinea pigs. The incidence of identical biotypes that were recovered repeatedly from the feces of the same animal was much higher in degraded carrageenan-treated guinea pigs than in the conventional untreated animals (table 3). Thus, colonization was much more common in the degraded carrageenan-treated groups. The exposure to contamination with gram-negative bacterial species from environmental sources, which, in these experiments, was primarily from the drinking water, was similar in the conventional degraded carrageenantreated and nontreated guinea pigs (table 2) .
At necropsy, the concentration of aerobic gram-negative bacteria in the duodenum and colon was only slightly higher in 2% degraded carrageenan-treated guinea pigs than in the nontreated controls; but it was substantially higher in the animals which received 5% degraded carrageenan in the drinking water (table 5) . However, 6 of the 10 2% degraded carrageenan-treated animals had been off treatment and had been maintained on ascorbic acid-supplemented tap water for 7 to 10 days before the time of necropsy and sampling of duodenal and colonic contents.
Discussion The ulcerative disease induced in conventional guinea pigs treated with de-
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VANDER WAAIJ ET AL. BIOTYPE CODE NO.
BACTERIAL SPECIES
Enterobocter cloacae
886271 624063 624127 568831
Klebsiella pneumoniae
560639 Enterobocter oerogenes
525759
Escherichia coli
723391 723389 723387 723381 526613
Pseudomonas oeruginaso
312064 303872 0
2 4
6
8
10 12 14 16 18
NUMBER OF ANIMALS COLONIZED
FIG. 7. Incidence of the various colonizing biotypes in conventional guinea pigs treated with degraded car· rageenan. 5. Concentration of aerobic gram-negative bacteria in the duodenum and colon at necropsy
TABLE
D egrad ed car· rageenan m wa ter
No. of anim a ls
Mean log concentration and range of gram-negative bacteria Duodenum
Colon
0 0.3 (0- 2) 3.0 (2- 4)
2.2(1 - 3) 2.5 (0- 5) 4.0 (2-5 )
'if
0 2
5 10
5
7
graded carrageenan was significantly more severe than in animals which were free of Enterobacteriaceae species. Accordingly, these results extend existing information about the guinea pig model of human inflammatory bowel disease, 14 and implicate the aerobic gram-negative intestinal microf1ora in the pathogenesis of the leswns. The gross and microsopic lesions in the large intestine (table 4) closely resembled those described by Watt and Marcus. 3 However, the striking increase in the number of cecal and colonic submucosal lymphoid patches (fig. 2B) that was observed in most of the conventional 2 and
5% degraded carrageenan-treated guinea pigs has not been mentioned in previous reports. Hyperplasia is a nonspecific reaction of gut-associated lymp hoid tissue to antigen usually originating in the bowel lumen . In germ-free guinea pigs, this lymphoid tissue is inapparent or absent and does not have germinal centers. 15 The same is true in other germ-free species. In the presence of degraded carrageenaninduced damage to the mucosal barrier of the large intestine, potentially pathogenic bacteria, such as Enterobacteriaceae species, may possibly enter the lamina propria or lymphatics and stimulate both submucosal lymphoid nodules and mesenteric lymph nodes. The presence of mesenteric lymphadenopathy as well as large intestinal submucosal lymphoid hyperplasia in the conventional guinea pigs (table 4) supports this interpretation. Furthermore, if antigens are shared by Enterobacteriaceae and the large bowel mucosa, as has been suggested in ulcerative colitis patients infected with E. coli (0119: B14), 8 the guinea pigs' immunological contact with these organisms may also contribute to ulcer formation.
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
Trimethoprim with sulfamethoxazole proved to be highly effective for selective decontamination of the digestive tract. This agent has been used in decontaminating human kidney transplantation patients, 16 and both trimethoprim with sulfamethoxazole and nalidixic acid have proved useful for selective decontamination of the rabbit intestinal tract (P. J. Heidt, in preparation). Strict isolation of the guinea pigs also was necessary to prevent recontamination after the Enterobacteriaceae species had been eliminated. By using plastic isolators under germ-free conditions, 12 it was possible to maintain 6 animals free of aerobic gram-negative organisms during the 30- or 37-day periods of degraded carrageenan administration. However, these guinea pigs were not germfree. A few unidentified gram-positive bacillus species were isolated, as well as the yeast, Candida albicans. These organisms are not sensitive to trimethoprim with sulfamethoxazole. The increase in the fecal concentration of Enterobacteriaceae species during degraded carrageenan treatment (table 3) most probably is related to a decrease in the colonization resistance. In mice, an increased fecal concentration of Enterobacteriaceae is a direct indicator of decreased colonization resistance. 10 In the conventional guinea pigs there was a 4-fold increase in the number of biotypes isolated repeatedly during treatment with degraded carrageenan (table 3); this is indicative of an increase in colonizations. A dependent relationship between the Enterobacteriaceae concentration and colonization resistance is further suggested by the finding of a relatively constant level of gram-negative bacteria in the drinking water (table 2). An increase in the number of colonizations during a period of unchanged resistance to colonization could only be explained by an increased daily oral contamination . This did not occur. Thus, it appears that resistance to bacterial colonization of the digestive tract was influenced directly or indirectly by degraded carrageenan, leading to a decreased colonization resistance and an increase in
471
the fecal concentration of Enterobacteriaceae species. In other animal species, increases in the concentration of intestinal bacteria are associated with invasion of these bacteria into the intestinal mucosa and lymph nodes"; it seems probable that the degraded carrageenan-treated conventional guinea pigs were similarly affected. The persistence of C. albicans, and its colonization in high concentration in the intestinal tract of the two 5% degraded carrageenan-treated Enterobacteriaceaefree animals is another indicator of reduced colonization resistance induced by degraded carrageenan . This may explain the presence of lesions such as cecal hemorrhages, and an occasional small cecal or colonic ulcer in these animals. However, the lesions were far less severe than in the conventional degraded carrageenantreated animals. The growth of C. albicans might have been prevented if an antifungal agent such as nystatin had been added to the drinking water. The results of the biotyping (table 3, fig. 7) support the interpretation that the family of Enterobacteriaceae species generally, and not a single specific biotype, was involved in the pathogenesis of ulcerative lesions in the conventional guinea pigs. However, the possibility that other potentially pathogenic microorganisms such as C. albicans may also play a role in large intestinal ulcerative disease induced by degraded carrageenan cannot be ruled out. Additional experiments are planned in germ-free or selectively decontaminated animals to further evaluate the pathogenosis of lesions in the absence of specific components of the microflora. The precise mechanism by which degraded carrageenan produces large bowel ulcerations remains unclear. 17 Watt and Marcus 4 have indicated that, in the rabbit, large bowel lesions are dose-related; fewer animals were affected and less severe lesions were noted, with a longer development time, at lower concentrations of degraded carrageenan. In our study, 5% degraded carrageenan produced more extensive lesions than did the 2% solution, although net intake of degraded carragee-
472
VANDER WAAIJ ET AL.
nan was about the same (table 3) . This suggests that degraded carrageenan may have a stronger direct effect on large bowel epithelium when the animals are partially dehydrated. Our results suggest a need for study of the possible effect(s) of degraded carrageenan on the secretory immune system (lgA) of the intestinal tract. IgA is known to be important in maintaining colonization res istance , largely by preventing bacterial adherence to the mucosa. 18 · 19 If this system is impaired, the colonization resistance decreases, thus allowing Enterobacteriaceae to adhere to the mucosa and enter the tissue, even in the absence of preexisting lesions . 20 The lymphoid hyperp lasia and mesenteric lymphadenopathy we observed in conventional guinea pigs treated with degraded carrageenan may h ave been nonspecific, but it could also have resulted from bacterial invasion of the mucosa as a result of the interference of de graded carrageenan with normal IgA act ivity. Additional studies are planned to identify the possible immunological mechanism(s) which may play a role in the experimentally induced disease. These studies may shed light on possible immunological factors affecting inflammatory bowel disease in human patients. REFERENCES 1. Wa tt J, Marcus R: Ulcerative colitis in the guinea pig caused by seaweed extract. J Pharm Pharmacal 21 (suppl):1875-1885, 1969 2. Marcus R, Watt J : Seaweeds and ulcerative colitis in laboratory animals . Lancet 2:489-490, 1969 3. Watt J , Marcus R: Carrageenan induced ul ceration of the large intestine in the guinea pig. Gut 12:164- 171, 1971 4. Watt J , Marcus R: Progress Report, Experimental ulcerative disease of the colon in animals. Gut 14: 506-510, 1973 5. Benitz KF, Goldberg L, Coulston F: Intesti nal effects of carrageenans in the rhesus monkey (Macaca mulatta). Fd Cosmet Toxicol 11 :565-575, 1973 6. Grosso P, Sharatt M , Carpanini FMB, et al:
7.
8.
9. 10.
11.
12.
13.
14.
15.
16.
17. 18.
19 .
20.
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Studies on carrageenan and large bowel ulceration in mammals. Fd Cosmet Toxicol11: 555-564, 1973 Watson DW, Quigley A, Bolt RJ: Effect of lymphocytes from patients with ulcera tive colitis on human ad ult colon ep ithelial cells. Gastroenterology 51:985-993, 1966 Shorter RG , Cardoza M , Huizenga KA, et al: Further studies of in vitro cytotoxicity of ly mphocytes for colonic epithelial cells . Gastroenterology 57: 30-35, 1969 Broberger 0: Immunologic studies in ulcerative colitis. Gastroenterology 47:229-240, 1964 van der Waaij D , Berghuis JM: Determination of coloni zation res istance of the di gestive tract of indi vidual mice. J Hyg (Camb) (i n press), van der Waaij D , Berghuis JM , Lekkekerk JEC: Colonization resistance of the digestive tract and the spread of bacteria to the lymphat ic organ s in mi ce. J Hyg (Camb) 70:335-342, 1972 Trexler PC, Reynolds LI : Flexible film apparatus for rearing and use of germ fre e animals. Appl Microbial 5:406- 410, 1957 van der Waaij D , Speltie TM, Vossen JM: Biotyping of Enterobacteriaceae as a test for the evaluation of isolation systems. J Hyg (Ca mb ) 70:639- 650, 1972 Mottet NK: On animal models for inflammatory bowel disease. Gastroenterology 62:1269- 1271, 1971 Sprinz H, Kundel DW, Dammin GJ eta!: The response of the germ free guinea pig to oral bacterial challenge with Escherichia coli and Shigella flexneri, with special reference to lymphatic tissue and the intestinal tract. Am J Pathol 39:681691, 1961 Dankert J: Biotypi ng of Enterobactericeae ; a method to determine t he efficiency of the barrier function of isolation units. In Germ free Research. Edited by JB Heneghan . New York, Academic Press, 1973, p 59-67 DiRosa M: Biological properties of carrageenan. J Pharm Pharmacal 24:89- 102, 1972 Freter R: Parameters affecting the assoc iation of vibrios with the intestinal surface in experimental cholera. Infect Immunol 6:134- 141 , 1972 Williams RC , Gibbons RJ: Inhibition of bacterial adherance by secretory immunoglobulin. Science 177:697- 699, 1972 van der Waaij D , Berghuis-deVries JM: Selective elimination of Enterobacteriaceae species from the digestive tract in mice and monkeys. J Hyg (Camb) (in press)
Vol. 67 . No. :3
GASTROENTEROLOGY
Copyri ght ©
197~
Printed in U.S.A.
MITIGATION OF EXPERIMENTAL INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS BY SELECTIVE ELIMINATION OF THE AEROBIC GRAM-NEGATIVE INTESTINAL MICROFLORA D. VAN DER WAAIJ, M.D., PH .D ., B. J. COHEN, D.V.M. , PH.D. , AND M . R. ANVER D.V .M ., PH.D. Unit for Laboratory Animal Medicin e, University of A1ichiga n , Ann Arbor, Michigan
Experimentally induced ulcerative disease of the large intestine in guinea pigs was significantly mitigated by selective elimination of the aerobic gram-negative intestinal microflora. Lesions were induced with 2 or 5% degraded carrageenan administered in the drinking water for 30 to 44 days. Craterous cecal or colonic ulcers, crypt abscesses, mucosal distorti on, mesenteric lymphadenopathy, marked cecal or colonic lymphoid hyperplasia , and other lesions were seen in conventional guinea pigs treated with degraded carrageenan . Other guinea pigs that were treated with 2% degraded carrageenan, but freed of Enterobacteriaceae species by the administration of trimethoprim with sulfamethoxazole, and subsequently maintained in isolators, exhibited virtually no lesions. Enterobacteriaceae-free animals that were treated with 5% degraded carrageenan had significantly fewer lesions than were observed in the conventional degraded carrageenan-treated animals. Biotyping of bacterial isolates indicated that the family of Enterobacteriaceae species generally, and not a single biotype, was associated with the lesions. The bacteria presumably stimulated an immunological response after penetration of the intestinal mucosal barrier ; thus , inflammatory bowel disease in guinea pigs, induced with degraded carrageenan , may provide a useful model for investigating immunological aspects of human inflammatory bowel disease. rhesus monkeys. t- s Initially, Watt and Marcus 1 induced ul cerations of the colon in guinea pigs that were fed 1% aqueous extracts of the dried red seaweed, Euchema spinosum, for 5 months. Subsequently, similar ulcerative lesions were produced within 30 days with 1 to 5% aqueous solu-
In recent years, ulcerative disease of the large intestine has been induced experimentally in several animal species, including guinea pigs, rabbits, rats, mice, and Received December 17, 1973. Accepted February 26, 1974 . Address requests for reprints to: Dr. Bennett J. Cohen, Unit for Laboratory Animal M edicine, 010 Animal Research Facility, University of Michi gan , Ann Arbor, Michiga n 48104. This investigation was s upported in p art by grants from the Anim al Resources Branch, Di vision of Research Resources, National Institutes of Health (RR 00200), from the University of Michigan Cancer Research Committee, and from National Science Foundation Institutional Grant (GU-3470). Dr. van der Waa ij's present address is: Radiobiological Institute, TNO, 151 Lange Kleiweg, Rij swijk (ZH), The Netherlands . The degraded carrageenan used in this study was
generously provided by Dr. G. B. Shirlaw and Dr. J. Thomas, Laboratoires Glaxo, S. A., Paris, Fra nce. The technical assistance of J ong Soo Park is gratefully ack nowled ged. The guinea pigs were housed in facili ties that are fully accredited by the American Assoc iation for Accreditation of Laboratory, Animal Care (AAA· LAC), and were ma inta ined in accordance wi t h stan· d ards in the Guide for the Care and Use of Lab oratory Animals. Departm en t of Health, Education a nd Wei· fare publication (National Institutes of Hea lth ) 73-23, Revised 1972.
460
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
tions of carrageenan, derived from the same seaweed but degraded by mild acid hydrolysis. 2 • 3 The clinical and pathological features of the experimentally induced disease resembled those of human ulcerative colitis in many respects, thus providing a simple model for investigating ulcerative disease of the colon. Several investigators have recently indicated the involvement of Escherichia coli (0119: B14) in the pathogenesis of human ulcerative colitis. Watson et al. 7 demonstrated that circulating lymphocytes, but not sera, from ulcerative colitis patients are cytotoxic in vitro for human adult autologous and allogeneic colonic epithelial cells. Shorter et al. 8 induced cytotoxicity for allogeneic colonic epithelial cells by preliminary incubation of lymphocytes derived from peripheral blood of normal healthy adults, with a lipopolysaccharide extract from E. coli (0119:B14). They suggested that the colonic ulcerations, with the resulting massive exposure of the individual to microorganisms from the lumen, may be an important factor in the induction of the cytotoxic property of the lymphocytes. Furthermore, these investigators speculated that this cytotoxicity may be introduced through shared antigenicity between bacteria and tissue constituents which may not be confined to E. coli (0119:B14). Thus, E. coli in particular, and Enterobacteriaceae species and other potentially pathogenic microorganisms in general, may play a significant role in the pathogenesis of ulcerative colitis. Ulcerative colitis patients often have a history of a bacterial infection of the large bowel. 8 • 9 The infection is always associated with bacterial invasion of the intestinal mucosa and an increase in the concentration of the causative bacterium in the feces. This suggests the possibility that a decrease in the colonization resistance of the digestive tract, which is also associated with an increase in concentration of potentially pathogenic intestinal bacteria, 10 is a factor in the pathogenesis of the lesions of ulcerative colitis. However, bacterial invasion of the intestinal mucosa may occur under a variety of conditions. For example, a high fecal concentration of a specific
461
bacterium has been demonstrated in mice, with subsequent passage of the bacterium through the intestinal mucosa into the mesenteric lymph nodes and spleen, after massive oral contamination with the bacterium. 11 The principal objective of this investigation was to determine the possible involvement of aerobic gram-negative intestinal bacteria in the pathogenesis of large intestinal ulcerations induced in guinea pigs with degraded carrageenan. We also attempted to link the occurrence of lesions with one or more specific bacteria, such as E. coli. Finally, we sought .to determine whether the administration · of degraded carrageenan to guinea pigs affects colonization resistance and the concentration of the aerobic gram-negative int~stinal microflora. Experimental Procedures Animals
Thirty-five male, English short-hair, albino guinea pigs were used. The animals were 3 to 7 months old and weighed 400 to 820 g. Each guinea pig was weighed twice weekly throughout the study . Housing and Care
Twenty-nine of the guinea pigs were housed under conventional conditions in a separate room in individual stainless steel cages equipped with grid floors. Six guinea pigs which were freed of Enterobacteriaceae, but which were not otherwise "germ free ," were maintained in identical cages or in clear plastic "shoebox" cages inside flexible plastic isolators. 12 The isolators were sterilized with 2% peracetic acid before the guinea pigs were introduced into them. The guinea pigs were fed a commercially available pelleted ration ad libitum. The diet was autoclaved at 121 C for 20 min to prevent any possibility of bacterial contamination. The drinking water was provided daily in bottles equipped with stainless steel sipper tubes . Ascorbic acid was added (0 .2 mg per ml) to compensate for the loss of vitamin C in the autoclaved diet. The drinking water also was used as the vehicle both for degraded carrageenan and the agent used to eliminate the Enterobacteriaceae from the intestinal flora; accordingly, sucrose (0 .05 g per ml) also was added to increase palatability.
462
VANDER WAAIJ ETAL.
Degraded carrageenan (Laboratoires Glaxo, Paris, France) was administered to conventional and Enterobacteriaceae-free guinea pigs in a 2 or 5% solution in the drinking water for 30 to 44 days (table 1) . The solutions were prepared fresh daily for the conventional guinea pigs. For the Enterobacteriaceae-free guinea pigs , stock solutions were prepared twice weekly under strict aseptic conditions using sterilized water. The guinea pigs were supplied daily from the stock solutions which were stored in the isolators. Trimethoprim with sulfamethoxazole, administered as Bactrim (Hoffmann-La Roche, Nutley , N.J.) was added to the stock solutions (0.25 mg per ml) to prevent multiplication of Enterobacteriaceae in the event of accidental contamination inside the isolators.
Microbiological Methods Elimination of Enterobacteriaceae species from the guinea pigs' intestinal microflora. Fresh feces from a group of 15 guinea pigs were cultured initially to determine the presence and concentration of aerobic gram-negative bacteria. Ten of these guinea pigs were shown to be free of Pseudomonas aeruginosa, which is resistant or has a low sensitivity to trimethoprim with sulfamethoxazole. However, this agent is highly effective against the Enterobacteriaceae. The Enterobacteriaceae flora of the 10 Pseudomonas-free guinea pigs was investigated for its sensitivity to trimethoprim with sulfamethoxazole by plating 6-hr broth cultures of fresh feces on Endo agar (Difco, Detroit, Mich.). Sensitivity discs (25, 50, 100 llg per disc) were placed on the plates before they were incubated overnight at 37 C. The Enterobacteriaceae microflora of the 10 TABLE
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guinea pigs was shown to be sensitive to trimethoprim with sulfamethoxazole, as indicated by an inhibition zone of more than 10 mm around all of the corresponding sensitivity discs for each animal. These guinea pigs then were transferred into a separate room where special precautions were taken to prevent contact contamination. Trimethoprim with sulfamethoxazole was administered in the drinking water (2 mg per ml) for 4 days, at which time 7 of the 10 guinea pigs were free of Enterobacteriaceae, as demonstrated by negative fecal cultures. These animals were then transferred into three isolators in groups of 2 or 3, and administration of trimethoprim with sulfamethoxazole was con tinued for an additional 10 days. Fresh feces were cultured daily to determine whether the guinea pigs remained free of aerobic gram-negative bacteria. Bacteriological culturing. Feces. Fresh feces were collected daily. Samples weighing approximately 100 mg were suspended in 0.5 ml of brain heart infusion broth (Difco). Subsequent serial dilutions were made with 0.05-ml diluting loops (Flow Laboratories , Rockville, Md.) in a plastic tray containing rows of eight cups, each filled with 0.5 ml of brain heart infusion broth. The diluting loops were heat-sterilized between dilution steps. The trays were placed in a plastic container and covered with aluminum foi l to prevent dehydration. After incubation overnight at 37 C, the various dilutions were subinocu lated on Endo agar plates which were incubated overnight at 37 C. The concentration of Enterobacteriaceae and Pseudomonas in the feces was interpreted from the growth observed on these plates. Water . Water from the drinking bottles of the conventionally housed guinea pigs was sampled
1. Experimental groups and treatments
No. examined Condition of guinea pigs
Degraded carrageenan treatm ent
Total no. of animals
Bacteriologically Daily feca l Enterobacteriaceae
Histologically Biotyping
concentration
Conventional Conventional Free ofEnterobacteriaceae and Pseudomonas Total
Controls; no degraded carrageenan administration 2% for 30- 44 days 5% for 30 days 2% for 37 days 5% for 30 days
9
5
5
9
13 7 4 2
10 7
10 7
13 7 4 2
35
28
22
35
4 2
September 1974
463
INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
before and after rinsing and refilling, and was cultured quantitatively in the same way as the fecal suspensions. Subinoculations on Endo agar were performed to determine the original concentration of gram-negative bacteria in the water samples, also in the same way as for the fecal suspensions. Biotyping. Once weekly a sample of fresh feces from each guinea pig was streaked directly on Endo agar and cultured in brain heart infusion broth. After 6 and 24 hr of incubation at 37 C, the broth cultures were subinoculated on Endo agar. The culture plates were examined after incubation overnight at 37 C. As many different looking colonies as possible from each plate were selected for subinoculation, with a minimum of 12 per sample. The colonies were subinoculated on other Endo agar plates to obtain pure cultures. The cultures thus obtained were subinoculated on Kligler agar slants (Difco) to further assure purity. Finally, the pure cultures were suspended in sterile saline, and these suspensions were used to inoculate sets of a commercially available series of biochemical and cultural tests (API 20 Enteric, Analytab Inc ., New York, N. Y.). This test series is useful in differentiating Enterobacteriaceae and other aerobic gram-negative bacteria. After inoculation, the test strips were incubated for 24 hr at 37 C, and the various reactions were interpreted in accordance with a key which is provided with the strips. The principles of this technique were identical to a previously described technique for biotyping Enterobacteriaceae, 13 although not all of the biochemical tests were identical. The results of the biochemical tests were coded by means of a binomial system which yielded an identification number for each of the various biotypes. 13 Arbitrary digital numbers were assigned in sequence at the time the 12 or more colonies were selected for biotyping. A graph was constructed to aid in determining whether the selection of 12 or more than 12 colonies was needed to assure an optimal inventory of the biotypes in each test sample (fig. 1). The abscissa of the graph described the number of morphologically different colonies typed; the ordinate identified the number of different biotypes in each group of four colonies. The 12 colonies originally selected for typing were divided arbitrarily into three sequential groups of four. The number of different biotypes in the first group of four was plotted , as illustrated in figure 1. The number of biotypes in the second group of four colonies that differed from those found in the first group of colonies, and from each other, also was plotted. This process was
q·· . . ~
f:::r--6 Sample of 5/14/73 o ....
o
Sample o f 6/4/73
~ 8
12
16
NUMBER OF MORPH OLOGIC ALLY DIF FERENT COLONIES TYPE D
FIG. 1. The number of colonies whose biotypes must be determined to assure an optimal inventory of each test sample. The curves derived from two tests on 1 guinea pig are shown. The results indicate that the biotyping of 12 colonies provided a complete inventory.
continued until the different biotypes in all three groups were similarly plotted. A "best fitting" curve was then drawn through the points. The intersection of this curve with the abscissa indicated the minimum number of colonies on which biotyping was needed to identify virtually all of the biotypes in the test sample . Preliminary tests, as illustrated in figure 1, indicated that the selection of 12 colonies usually was adequate to provide this inventory. Thus, 12 colonies were routinely biotyped; if, however, the curve did not reach the abscissa at the third group of colonies, additional colonies were selecte d for biotyping from the original Endo agar plates that had been stored at 4 C.
Necropsy and Histological Methods At the end of the experimental period each guinea pig was killed with C0 2 gas and necropsied immediately. The thoracic and abdominal viscera were examined grossly. Sections of the various organs were fixed in 10% buffered neutral formalin. The gastrointestinal tract was removed in its entirety. The cecum and colon were emptied of feces and inspected for gross evidence of ulcerations or other lesions. Stomach, duodenum, jejunum, ileum, cecum, proximal colon, midcolon, and distal colon also were fixed in formalin for subsequent histological examination. Fixed tissues were embedded in paraffin, sectioned at 5 !J. , stained with hematoxylin and eosin, and examined routinely by light microscopy. For histochemical identification of pigment, representative sections of cecum, colon, mesenteric lymph nodes, and spleen were also stained with periodic acid-
464
VANDER WAAIJ ET AL.
Schiff, Sudan Black B for lipid , and Perls' Prussian blue for iron .
Results Clinical Course The conventional guinea pigs drank nearly twice as much of the 2% degraded carrageenan solution t han they did the 5% solut ion (table 2). As a result, the actual daily intake of degraded carrageenan was similar for both groups (tab le 3) . The intakes recorded for the Enterobacteriaceae-free animals were identical. A few guinea pigs refused the 5% solution and became severely dehydrated. These animals were removed from the experiment and are not included in the results. The clinical course of the conventional guinea pigs was similar to that described by Watt and Marcus. 3 Animals receivi ng 2 or 5% degraded carrageenan invariably exhibited weight loss or a sharpiy flattened growth curve starting within several days to 2 weeks after administration of degraded carrageenan was begun. The weight loss was greater in t he animals given 5% degraded carrageenan. All of the guinea pigs which received 5% degraded carrageenan, TABLE
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and many but not a ll of those that received the 2% solution, exhibited soft feces during the period of degraded carrageenan administration. Blood was grossly visible in the feces of 3 of the 7 animals which received the 5% solution, but in only 2 of 13 animals which received 2% degraded carrageenan. All 4 of the Enterobacteriaceae-free guinea pigs which received 2% degraded carrageenan gained weight throughout the period of administration and produced normally firm fecal pellets. The 2 Enteroba cteriacea-free guinea pigs which received 5 9C degraded carrageenan lost weight during the period of administration. Soft feces were noted sporadically, but during most of the period of degraded carrageenan administration these 2 animals produced firm but smaller than normal fecal pellets . Necropsy and Histologica l Findings Consistently found at necropsy in the conventional guinea pigs which received 2 or 5% degraded carrageenan were innumerable white foci of varying sizes that were visible grossly through the serosa of the cecum and colon (tab le 4, fig . 2B). The histological picture was that of nor-
2. Concentration of aerobic gram-negative bacteria in the drinking water of conventional guinea pigs Mean log concentration of gra m- nega ti ve
% Degraded
No . of
carrageenan
in the water
a nim als
0 2 5
5 10 7
bacteria/m l" Water intake" Just before refilling water bottl es
Directl v after refilling wa.ter bot tles
5.7 (4- 7) 6.3 (4-7) 6.0 (4-7)
:<; 1 :<; 1 :<; 1
ml/day
a
85.0 (68- 100) 74.0 (59-97) 38.5 (30- 44)
Numbers in parentheses a re range.
TABLE
3. Daily dose of degraded carrageenan and colonization properties of aerobic gram-negative bacteria isolated from fec es of conventional guinea pigs Mean and range of:
Degraded carrageenan in water
No. of animals
Body wt at start of experiment
carragee n an
Log conce ntration of gram-negative
No . of hioty pes isola ted once
No. identical biotypes isolated
1.4 (0-4) 2.7 (1-5) 0.1 (0-1)
1.0 (0. 2) 4.1 (3.9- 5.4) 4.0 (3- 5)
bacteria/g of feces
repeated!~·
g/kg body wt
%
0 2 5
Da ily dose of degrad ed
5 10 7
780 (71 5-870) 550 (500-610) 820 (740-920)
2.5 (2.1 -3.3 ) 2.4 (1.7- 2.8)
1.7 (1.5-1.9) 4.1 (3.9-5.4) 4.5 (4.0-4.9)
September 1974 TABLE
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INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
4. L esions in conventional and Ent erobacteriaceae-free guin ea pigs treated with degraded carrageenan Enterobacteriaceae·free
Conventional Treated with degraded ca rrageenan
Lesion
Treated with d egraded ca rrageenan
Unt reated
In creased cecal or colon ic submu cosal lymphoid tissue Enlarge ment of cecal or co lonic ly mph nodes Crypt abscesses Cec um Distal co lon Ulcers Cecum Distal colon Polymorp honuclear leukocytes in lam in a propria Cecum Distal colon Mucosal di stortion Cec um Dista l colon Mucosal a nd submucosal gra nulation tissue Cec um Distal co lo n Mesenteri c fibros is
2%30-44 days
5% 30 days
2%37 days
5% 30 days
1/ 9"
11/ 13
7/7
1/ 4
1/2
0/ 9
3/13
6/7
0/ 4
2/2"
0/ 9 0/7
1/ 13 1/13
5/7 4/7
0/ 4 0/4
1/2 0/ 2
0/9 0/7
3/13 5/13
2/7 4/7
0/4 0/4
1/2 1/2
2/9 2/7
8/13 5/ 13
4/ 7 5/7
0/4 0/ 4
2/2 0/ 2
0/ 9 0/7
2/13 5/13
4/7 4/7
0/ 4 0/ 4
1/2 1/2
0/ 9 0/7 0/ 9
4/ 13 5/13 1/ 13
4/7 4/7 4/ 7
0/ 4 0/4 0/4
1/2 1/2 0/2
"No. with les ions per total of no. exam in ed. • Slight e nl a rge ment.
mally active submucosal lymphoid tissue; but the number of such foci was far greater than in conventional untreated guinea pigs (fig. 2A). Two of the degraded carrageenan-treated Enterobacteriaceae-free guinea pigs also exhibited a moderate increase in cecal or colonic lymphoid patches, and other lesions (table 4) also were present. However, this group of guinea pigs generally was more comparable to untreated controls than to degraded carrageenan-treated conventional animals. Typical findings (table 4) in the degraded carrageenan-treated conventional guinea pigs were: enlarged cecal or colonic lymph nodes, cecal or colonic mucosal crypt abscesses and mucosal distortion (fig. 3), gross and microscopic craterous ulcers (figs. 4 and 5), polymorphonuclear leukocytes in the lamina propria, mucosal and submucosal granulation tissue, and fibrosis of the mesentery (fig. 6). The lesions were
more extensive and occurred more frequently in the guinea pigs treated with 5% degraded carrageenan than in those treated with the 2% solution. The ulcers in these conventional guinea pigs also were more severe and widespread in the distal colon than in the cecum . Ulcers were not seen in the proximal colon. In general, the ulcerative lesions closely resembled those described by Watt and Marcus . 3 In addition, dense deposits of iron-positive pigment, interpreted as hemosiderin, were demonstrated consistently in the submucosa of the proximal colon and cecum, but only infrequently in the distal colon, both of conventional and Enterobacteriaceae-free guinea pigs which had received degraded carrageenan . The pigment was similarly present, but to a much lesser extent, in conventional untreated animals. In 4 of the 6 degraded carrageenan-treated Enterobacteriaceae-free guinea pigs, focal
466
VANDER WAAIJ ET AL.
hemorrhages were present m the lamina propria or submucosa. Microbiological Results The feces of the guinea pigs in the isolators remained free of Enterobacteriaceae species and of Pseudomonas aeruginosa throughout the experiment.
Vol. 67, No. 3
The only aerobic microorganisms isolated from these animals were a few unidentified bacillus species and a yeast, Candida albicans. Candida was present in a concentration of 10 4 to 10 8 per g of feces in the two 5% degraded carrageenan-treated Enterobacteriaceae-free animals . Two of the 2% degraded carrageenan-treated En-
FIG. 2. A, cecum of a n unt reated, control guinea pig with a few , scattered lymphoid foci ( x 1.5). B, multiple white ly mphoid foci see n through the cecal serosa in a 5% degraded carrageenan-treated gu inea pig. Compare with A ( x 0.5) .
-111111 .11 -llllllllll t II
II JIll I II1111111 1' Ill t-lllll !11 111111 !II II
FIG. 4. Distal colon mucosa of 5% degraded carrageenan-treated animal with multiple ulcers ( x 3).
467
468
VANDER WAAIJ ET AL.
terobacteriaceae-free animals were negative for Candida, while 2 had fecal concentrations up to 10 4 per g. In the conventional guinea pigs, yeasts were only occasionally found, and only in concentrations less than 10 2 per g. In the conventional guinea pigs, the concentration of Enterobacteriaceae species in the feces invariably increased within
Vol. 67, No. 3
the first 3 days, and remained high throughout the degraded carrageenan treatment period (table 3). These animals were colonized with a relatively small num· her of different biotypes (fig. 7). Six different klebsiella biotypes were isolated, one of which was found in all 17 animals, while another occurred in 7 animals; the remain· ing four biotypes occurred less frequently.
FIG. 5. Chronic ulcer in distal colon of 5% degraded carrageenan·treated animal. Calcification (large arrow) and fibrosis at base of ulcer with masses of overlying polymorphonuclear leukocytes . Muscularis mucosae (small arrow) intact; submucosa contains many foamy macrophages and polymorphonuclear leukocytes (hematoxylin and eosin. x 430).
September 1974
INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
469
FIG. 6. Fibrosis and foamy macrophages in the mesentery of an ulcerated distal colon from a guinea pig treated with 5% degraded carrageenan (hematoxylin and eosin, x 100) .
E. coli was represented by five different biotypes, while only two different biotypes of Pseudomonas aeruginosa were isolated in repeated weekly samples from 2 of the 17 guinea pigs. The incidence of identical biotypes that were recovered repeatedly from the feces of the same animal was much higher in degraded carrageenan-treated guinea pigs than in the conventional untreated animals (table 3). Thus, colonization was much more common in the degraded carrageenan-treated groups. The exposure to contamination with gram-negative bacterial species from environmental sources, which, in these experiments, was primarily from the drinking water, was similar in the conventional degraded carrageenantreated and nontreated guinea pigs (table 2) .
At necropsy, the concentration of aerobic gram-negative bacteria in the duodenum and colon was only slightly higher in 2% degraded carrageenan-treated guinea pigs than in the nontreated controls; but it was substantially higher in the animals which received 5% degraded carrageenan in the drinking water (table 5) . However, 6 of the 10 2% degraded carrageenan-treated animals had been off treatment and had been maintained on ascorbic acid-supplemented tap water for 7 to 10 days before the time of necropsy and sampling of duodenal and colonic contents.
Discussion The ulcerative disease induced in conventional guinea pigs treated with de-
470
Vol . 67, No. 3
VANDER WAAIJ ET AL. BIOTYPE CODE NO.
BACTERIAL SPECIES
Enterobocter cloacae
886271 624063 624127 568831
Klebsiella pneumoniae
560639 Enterobocter oerogenes
525759
Escherichia coli
723391 723389 723387 723381 526613
Pseudomonas oeruginaso
312064 303872 0
2 4
6
8
10 12 14 16 18
NUMBER OF ANIMALS COLONIZED
FIG. 7. Incidence of the various colonizing biotypes in conventional guinea pigs treated with degraded car· rageenan. 5. Concentration of aerobic gram-negative bacteria in the duodenum and colon at necropsy
TABLE
D egrad ed car· rageenan m wa ter
No. of anim a ls
Mean log concentration and range of gram-negative bacteria Duodenum
Colon
0 0.3 (0- 2) 3.0 (2- 4)
2.2(1 - 3) 2.5 (0- 5) 4.0 (2-5 )
'if
0 2
5 10
5
7
graded carrageenan was significantly more severe than in animals which were free of Enterobacteriaceae species. Accordingly, these results extend existing information about the guinea pig model of human inflammatory bowel disease, 14 and implicate the aerobic gram-negative intestinal microf1ora in the pathogenesis of the leswns. The gross and microsopic lesions in the large intestine (table 4) closely resembled those described by Watt and Marcus. 3 However, the striking increase in the number of cecal and colonic submucosal lymphoid patches (fig. 2B) that was observed in most of the conventional 2 and
5% degraded carrageenan-treated guinea pigs has not been mentioned in previous reports. Hyperplasia is a nonspecific reaction of gut-associated lymp hoid tissue to antigen usually originating in the bowel lumen . In germ-free guinea pigs, this lymphoid tissue is inapparent or absent and does not have germinal centers. 15 The same is true in other germ-free species. In the presence of degraded carrageenaninduced damage to the mucosal barrier of the large intestine, potentially pathogenic bacteria, such as Enterobacteriaceae species, may possibly enter the lamina propria or lymphatics and stimulate both submucosal lymphoid nodules and mesenteric lymph nodes. The presence of mesenteric lymphadenopathy as well as large intestinal submucosal lymphoid hyperplasia in the conventional guinea pigs (table 4) supports this interpretation. Furthermore, if antigens are shared by Enterobacteriaceae and the large bowel mucosa, as has been suggested in ulcerative colitis patients infected with E. coli (0119: B14), 8 the guinea pigs' immunological contact with these organisms may also contribute to ulcer formation.
September 1974
INFLAMMATORY BOWEL DISEASE IN GUINEA PIGS
Trimethoprim with sulfamethoxazole proved to be highly effective for selective decontamination of the digestive tract. This agent has been used in decontaminating human kidney transplantation patients, 16 and both trimethoprim with sulfamethoxazole and nalidixic acid have proved useful for selective decontamination of the rabbit intestinal tract (P. J. Heidt, in preparation). Strict isolation of the guinea pigs also was necessary to prevent recontamination after the Enterobacteriaceae species had been eliminated. By using plastic isolators under germ-free conditions, 12 it was possible to maintain 6 animals free of aerobic gram-negative organisms during the 30- or 37-day periods of degraded carrageenan administration. However, these guinea pigs were not germfree. A few unidentified gram-positive bacillus species were isolated, as well as the yeast, Candida albicans. These organisms are not sensitive to trimethoprim with sulfamethoxazole. The increase in the fecal concentration of Enterobacteriaceae species during degraded carrageenan treatment (table 3) most probably is related to a decrease in the colonization resistance. In mice, an increased fecal concentration of Enterobacteriaceae is a direct indicator of decreased colonization resistance. 10 In the conventional guinea pigs there was a 4-fold increase in the number of biotypes isolated repeatedly during treatment with degraded carrageenan (table 3); this is indicative of an increase in colonizations. A dependent relationship between the Enterobacteriaceae concentration and colonization resistance is further suggested by the finding of a relatively constant level of gram-negative bacteria in the drinking water (table 2). An increase in the number of colonizations during a period of unchanged resistance to colonization could only be explained by an increased daily oral contamination . This did not occur. Thus, it appears that resistance to bacterial colonization of the digestive tract was influenced directly or indirectly by degraded carrageenan, leading to a decreased colonization resistance and an increase in
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the fecal concentration of Enterobacteriaceae species. In other animal species, increases in the concentration of intestinal bacteria are associated with invasion of these bacteria into the intestinal mucosa and lymph nodes"; it seems probable that the degraded carrageenan-treated conventional guinea pigs were similarly affected. The persistence of C. albicans, and its colonization in high concentration in the intestinal tract of the two 5% degraded carrageenan-treated Enterobacteriaceaefree animals is another indicator of reduced colonization resistance induced by degraded carrageenan . This may explain the presence of lesions such as cecal hemorrhages, and an occasional small cecal or colonic ulcer in these animals. However, the lesions were far less severe than in the conventional degraded carrageenantreated animals. The growth of C. albicans might have been prevented if an antifungal agent such as nystatin had been added to the drinking water. The results of the biotyping (table 3, fig. 7) support the interpretation that the family of Enterobacteriaceae species generally, and not a single specific biotype, was involved in the pathogenesis of ulcerative lesions in the conventional guinea pigs. However, the possibility that other potentially pathogenic microorganisms such as C. albicans may also play a role in large intestinal ulcerative disease induced by degraded carrageenan cannot be ruled out. Additional experiments are planned in germ-free or selectively decontaminated animals to further evaluate the pathogenosis of lesions in the absence of specific components of the microflora. The precise mechanism by which degraded carrageenan produces large bowel ulcerations remains unclear. 17 Watt and Marcus 4 have indicated that, in the rabbit, large bowel lesions are dose-related; fewer animals were affected and less severe lesions were noted, with a longer development time, at lower concentrations of degraded carrageenan. In our study, 5% degraded carrageenan produced more extensive lesions than did the 2% solution, although net intake of degraded carragee-
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nan was about the same (table 3) . This suggests that degraded carrageenan may have a stronger direct effect on large bowel epithelium when the animals are partially dehydrated. Our results suggest a need for study of the possible effect(s) of degraded carrageenan on the secretory immune system (lgA) of the intestinal tract. IgA is known to be important in maintaining colonization res istance , largely by preventing bacterial adherence to the mucosa. 18 · 19 If this system is impaired, the colonization resistance decreases, thus allowing Enterobacteriaceae to adhere to the mucosa and enter the tissue, even in the absence of preexisting lesions . 20 The lymphoid hyperp lasia and mesenteric lymphadenopathy we observed in conventional guinea pigs treated with degraded carrageenan may h ave been nonspecific, but it could also have resulted from bacterial invasion of the mucosa as a result of the interference of de graded carrageenan with normal IgA act ivity. Additional studies are planned to identify the possible immunological mechanism(s) which may play a role in the experimentally induced disease. These studies may shed light on possible immunological factors affecting inflammatory bowel disease in human patients. REFERENCES 1. Wa tt J, Marcus R: Ulcerative colitis in the guinea pig caused by seaweed extract. J Pharm Pharmacal 21 (suppl):1875-1885, 1969 2. Marcus R, Watt J : Seaweeds and ulcerative colitis in laboratory animals . Lancet 2:489-490, 1969 3. Watt J , Marcus R: Carrageenan induced ul ceration of the large intestine in the guinea pig. Gut 12:164- 171, 1971 4. Watt J , Marcus R: Progress Report, Experimental ulcerative disease of the colon in animals. Gut 14: 506-510, 1973 5. Benitz KF, Goldberg L, Coulston F: Intesti nal effects of carrageenans in the rhesus monkey (Macaca mulatta). Fd Cosmet Toxicol 11 :565-575, 1973 6. Grosso P, Sharatt M , Carpanini FMB, et al:
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Studies on carrageenan and large bowel ulceration in mammals. Fd Cosmet Toxicol11: 555-564, 1973 Watson DW, Quigley A, Bolt RJ: Effect of lymphocytes from patients with ulcera tive colitis on human ad ult colon ep ithelial cells. Gastroenterology 51:985-993, 1966 Shorter RG , Cardoza M , Huizenga KA, et al: Further studies of in vitro cytotoxicity of ly mphocytes for colonic epithelial cells . Gastroenterology 57: 30-35, 1969 Broberger 0: Immunologic studies in ulcerative colitis. Gastroenterology 47:229-240, 1964 van der Waaij D , Berghuis JM: Determination of coloni zation res istance of the di gestive tract of indi vidual mice. J Hyg (Camb) (i n press), van der Waaij D , Berghuis JM , Lekkekerk JEC: Colonization resistance of the digestive tract and the spread of bacteria to the lymphat ic organ s in mi ce. J Hyg (Camb) 70:335-342, 1972 Trexler PC, Reynolds LI : Flexible film apparatus for rearing and use of germ fre e animals. Appl Microbial 5:406- 410, 1957 van der Waaij D , Speltie TM, Vossen JM: Biotyping of Enterobacteriaceae as a test for the evaluation of isolation systems. J Hyg (Ca mb ) 70:639- 650, 1972 Mottet NK: On animal models for inflammatory bowel disease. Gastroenterology 62:1269- 1271, 1971 Sprinz H, Kundel DW, Dammin GJ eta!: The response of the germ free guinea pig to oral bacterial challenge with Escherichia coli and Shigella flexneri, with special reference to lymphatic tissue and the intestinal tract. Am J Pathol 39:681691, 1961 Dankert J: Biotypi ng of Enterobactericeae ; a method to determine t he efficiency of the barrier function of isolation units. In Germ free Research. Edited by JB Heneghan . New York, Academic Press, 1973, p 59-67 DiRosa M: Biological properties of carrageenan. J Pharm Pharmacal 24:89- 102, 1972 Freter R: Parameters affecting the assoc iation of vibrios with the intestinal surface in experimental cholera. Infect Immunol 6:134- 141 , 1972 Williams RC , Gibbons RJ: Inhibition of bacterial adherance by secretory immunoglobulin. Science 177:697- 699, 1972 van der Waaij D , Berghuis-deVries JM: Selective elimination of Enterobacteriaceae species from the digestive tract in mice and monkeys. J Hyg (Camb) (in press)