parer leur reaction avec celle de chiots t6moins. Les chiots d6ve- lopperent une lassitude transi- toire et de l'inapp6tence, ainsi qu'une l6gere diarrhee et du.
Campylobacterjejuni Colitis in Gnotobiotic Dogs J.F. Prescott, I.K. Barker, K.I. Manninen and O.P. Miniats*
ABSTRACT Campylobacter jejuni of human and canine origin was inoculated orally into six gnotobiotically reared Beagle puppies and reactions were compared with two controls. Inoculated dogs developed transient lassitude, inappetence, mild diarrhea and tenesmus during the period 36-72 hours after inoculation. Pairs of dogs killed 43 hours, and five and seven days after inoculation had lesions limited to typhlitis and colitis. Congestion of colonic mucosa, associated loss of goblet cells, attenuation and exfoliation of surface epithelium with microerosions, hypertrophy of glands and neutrophil infiltration of lamina propria were seen during the acute phase. Less severe surface and inflammatory lesions were evident at five and seven days, with hyperplasia of the proliferative compartment in mucosal glands. Campylobacter established at over 1010 organisms per gram of colonic content but did not invade the mucosa. It was concluded that the gnotobiotic dog may be a suitable model for investigation of the pathogenesis of Campylobacter colitis.
lopperent une lassitude transitoire et de l'inapp6tence, ainsi qu'une l6gere diarrhee et du tenesme, de 36 a 72 heures apres l'infection. Les couples de chiots sacrifies au bout de 43 heures ou de cinq et sept jours apres l'infection, n'afficherent que de la typhlite et de la colite. Au cours de la phase aigue de la maladie, il se produisit de l'hyperemie de la muqueuse du colon, une perte de cellules a gobelet, de l'attenuation et de la desquamation epitheliales superficielles, accompagnees d'erosions microscopiques, une hypertrophie glandulaire et une infiltration du chorion par des neutrophiles. Les chiots sacrifies aux cinquieme et septieme jours apres l'infection afficherent des lesions superficielles et inflammatoires moins marquees; ces lesions s'accompagnaient d'une hyperplasie de la partie proliferative des glandes de la muqueuse. C. jejuni depassa 1010 organismes par gramme de contenu du colon, mais il n'envahit pas la muqueuse. Les resultats de cette experience permirent de conclure que les chiots gnotoxeniques pourraient representer un modele satisfaisant pour l'etude de la pathogenese de la colite imputable a C. jejuni.
RlSUM: Cette experience consistait A faire ingerer A six chiots Beagle gnotoxdniques, Campylobacter jejuni d'origine humaine et canine; elle visait aussi A comparer leur reaction avec celle de chiots t6moins. Les chiots d6ve-
INTRODUCTION Campylobacter jejuni has recently been recognized as a major cause of bacterial diarrhea in man (2, 3, 5, 24). The clinical manifestations have been well described (6, 13). The main site of infection
seems to be the lower small intestine, but the colon is frequently also involved (4, 14). The epidemiology of the disease is not fully understood, although it is clear that domestic animals are one reservoir of infection. Outbreaks of Campylobacter enteritis have been caused by bovine fecal contamination of milk (3, 23). Campylobacter jejuni is present in the feces of pigs (6, 18), of sheep, in which the organism is a cause of abortion (26), of dogs (1, 12, 24), and in large numbers of chickens (9). There is a clear association between diarrhea in puppies and C. jejuni infection in their owners (1, 24, 27). There is still debate, however, as to whether C. jejuni is a cause of diarrhea in dogs. Hosie et al (12) found no difference in prevalence of infection between normal and diarrheic dogs, and the descriptions of Campylobacter enteritis in dogs (17, 19, 25) are not adequate to rule out other known causes of diarrhea. Prescott and Karmali (21) were unable to produce diarrhea in puppies with large numbers of organisms of human origin. The object of the work described here was to attempt to produce Campylobacter enteritis in gnotobiotic puppies and to describe the changes associated with the infection. This work has been the subject of a preliminary report elsewhere (22).
MATERIALS AND METHODS DERIVATION AND REARING OF GNOTOBIOTIC PUPPIES
Two litters of Beagle puppies derived by Caesarian section
were
*Department of Veterinary Microbiology and Immunology (Prescott, Manninen), Department of Pathology (Barker) and Department of Clinical Studies (Miniats), Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada NlG 2W1. Submitted January 27, 1981. Can. J. comp. Med. 45: 377-383 (October 1981)
377
using standard techniques (11). Eight puppies were obtained and reared gnotobiotically together in a germ free isolator by methods described (11). Rectal swabs and isolators were monitored bacteriologically by routine techniques. EXPERIMENTAL PROCEDURES
Infecting organisms - Two C. jejuni isolates were used. An isolate from a case of diarrhea in man (human isolate, CFJ1) was obtained from Dr. M.A. Karmali (Hospital for Sick Children, Toronto, Canada) and stored in liquid nitrogen until used. An isolate from a diarrheic puppy (dog isolate, CFJ14) was obtained from Dr. M.J. Blaser (Microbiology Laboratory, Denver Veterans Administration Medical Center, Colorado), and similarly treated. Organisms were grown microaerophilically on blood agar at 42°C for 48 hours, washed with sterile phosphate-buffered saline and introduced in a sterile manner into the germfree isolators. Experimental design - The puppies were assigned to four pairs. The first pair consisted of two puppies transferred at 22 days of age into a separate isolator. Each was inoculated with 3.9 x 108 viable C. jejuni CFJ1, observed for one week and killed. The second pair were uninfected control puppies killed at 30 days of age. The remaining four dogs were then divided into two pairs and placed in separate germfree isolators. Those in one group were each inoculated with a total of 4.4 x 108 viable C. jejuni CFJ1 and those in the other with 5.2 x 1012 C. jejuni CFJ14. Dogs were observed following infection and one from each pair was killed at 43 hours and the other at five days after inoculation.
Observations Puppies were observed at intervals of four to six hours following infection. The fol-
lowing observations were recorded: rectal temperature, quality of feces, and the behavior of the puppies (desire to feed or sleep, presence of straining to pass feces, reaction to handling). Serum was 378
taken from puppies before inoculation and at death. Feces were examined by direct Gram stained smear for Campylobacter and other bacteria.
tured microaerophilically for Campylobacter and other bacteria: heart blood, liver, gall bladder, spleen, lung, tonsil, mesenteric lymph node and kidney.
Postmortem examination - Puppies were killed by an overdose of a barbiturate. Intestinal tracts were immediately removed and examined grossly. Sections of the cecum, colon, ileum, three parts of the jejunum, two parts of the duodenum, stomach and mesenteric lymph nodes were fixed in 10% buffered formalin, embedded in paraffin and sectioned at 6 ,um. Sections were stained with hematoxylin and eosin, Alcian blue, the Warthin-Starry silver stain, and by the Periodic acid Schiff reaction. Liver, lung, spleen, kidney, heart, tonsil and adrenals were similarly treated. Colonic mucosa was fixed in 4% glutaraldehyde phosphate buffer, pH 7.2. Thin sections were prepared by standard methods, and were examined with a Philips 200 transmission electron microscope.
Determination of humoral antibody titer - Antibody titers were determined in sera by means of the indirect fluorescent antibody test described by Blaser et al (3), using CFJ1 as the antigen. Positive control serum prepared in a dog by intravenous inoculations of CFJ1 consistently gave titers of 512 using either CFJ1 or CFJ14 as the antigen; positive serum was always included in the test as a control. Fluorescein isothiocyanate conjugated rabbit anti-dog IgG (1:16 dilution in 0.1% Amido black, Cappel Laboratories, Cochranville, Pennsylvania) was used. Slides were read as unknowns and titers judged by the methods described (2).
Bacteriological examination -The number of C. jejuni in the lumen of the colon and ileum were quantified by standard bacteriological methods. The number present in the duodenum, midjejunum and other organs were roughly quantified as described in Table I. The following tissues were also cul-
RESULTS No difficulty was experienced in rearing healthy gnotobiotic puppies; they were judged to be normally developed for their age. The puppies all became contaminated with two strains of Staphylococcus epidermidis, which reached large numbers in the gut. The two control puppies had larger numbers of staphylococci in their colons (8.8 x
TABLE I. Isolation of Campylobacter jejuni at Various Times after Inoculation Into Gnotobiotic Puppies Human Isolate (CFJ1) Time 7d 7d 43h 5d 2.5 x 10l°& 1.3 x 1010 1.7 x 1010 9.7 x 109 7.8 x 106 6.2 x 109 1.3 x 107 3.9 x 107 2+b 2+ 3+ 2+ 1+ 1+ 2+ 3+
Organ Colon Ileum Mid-jejunum Duodenum Heart blood 1+ 2+ Liver 3+ Gall bladder 1+ 3+ Spleen 1+ 3+ Lung 1+ 2+ Tonsil Mesenteric 1+ 1+ lymph node Kidney 'Counts per gram of content b1+, Campylobacter in first quadrant; 2+ quadrants of the culture plate
Dog Isolate (CFJ14) Time 5d 43h 6.1 x 1010 1.5 x 1010 2.0 x 108 3.5 x 108 3+ 3+ 3+ 3+ -
+
1+ 1+
3+
2+
2+ 3+
1+
2+ 1+
2+
-
2+
3+
-
-
2+ 3+
2+
1+ 1+ -
3+
-
-
2+ -
-
in 2nd quadrant; 3+, over the 3rd and 4th
109 and 2.1 x 1010 per gram of contents) than the C. jejuni infected puppies (a mean 4.6 x 108, range 1.1 x 108 to 1.1 x 109 per gram of contents). No other bacteria were detected. The effect on the puppies of infection with either CFJ1 or CFJ14 was similar. No increase in rectal temperature was observed. Between 36 and 43 hours after infection the puppies were observed to become quieter and less playful, to sleep more, and to drink about half the usual quantity of milk. This period of malaise was transient and lasted for 12-18 hours. During this period the feces were looser than at previous or subsequent times, and there was evidence of slight fecal staining around the anus. It was not possible to determine the number of fecal specimens passed since the feces of both uninfected and infected dogs were soft and passed in such small quantities that they were soon smeared and lost on the cage floor. No blood was distinguished in the feces, which at no time could be described as frankly diarrheic. Infected puppies were observed periodically to squat and to strain as though to pass feces, whining while they did so, but straining was usually unproductive. The period of straining lasted from about 36 hours to 72 hours after infection (a total of 36 hours). One of the puppies infected for seven days appeared to lose weight over this period. No gross changes were seen at necropsy in uninfected control dogs. On autopsy of the puppy infected for 43 hours with CFJ1, gross changes were confined to the intestine. The full length of the colon showed patchy congestion which was more severe distally (Fig. 1). The wall of the colon was edematous and the colonic contents abnormally fluid. The entire length of the small intestine appeared thickened, corrugated and slightly pink. The right and middle colic lymph nodes were congested. The intestinal tract of the puppy infected for 43 hours with CFJ14 showed similar but less marked changes in the colon,
Fig. 1. Congestion of colonic mucosa of a dog killed 43 hours after inoculation with
Campylobacterjejuni of human origin. X3.4.
although the small intestine appeared normal. The puppy infected for five days with CFJ1 showed slight congestion throughout the intestine; the contents of the colon were excessively liquid and gaseous. The puppy infected with CFJ14 for five days showed only several areas of congestion in the terminal rectum. The two puppies infected with CFJ1 for seven days showed no gross intestinal change. Significant microscopic lesions were limited to the ceca and colons of Campylobacter inoculated dogs, and no consistent difference was noted between animals inoculated with isolates of human or canine
origin.
The surface epithelium of these organs in both control dogs consisted of columnar cells with an obvious brush border and nuclei toward the middle or base of the cell (Fig. 2). Interspersed were goblet cells in a ratio of goblet: epithelial cells of about 1:5 to 1:10. Scattered coccoid bacteria were present over the surface. The neck of glands was composed mainly of goblet cells, with basal nuclei, while interspersed were columnar epithelial cells with eosinophilic cytoplasm. At the base of glands
and occupying the deepest 10-20% of the gland were cells with large vesiculate open nuclei, with prominent nucleoli. The cytoplasm of cells in this zone was moderately basophilic. Mitotic figures were limited to this area and were relatively infrequent, about one or two per gland. Mucus in goblet cells on the surface and upper portions of glands stained with both PAS and Alcian blue, while goblet cells deeper in glands tended to be more uniformly Alcian blue positive. Inflammatory cells in the lamina propria of control animals were uncommon and restricted to scattered lymphocytes in the superficial half of the mucosa and rare theliolymphocytes. Responses to infection varied between dogs examined at the same interval after inoculation, and changes tended to be patchy in distribution within the same dog. By 43 hours after inoculation, the mucosal thickness in infected dogs had increased substantially and this change persisted throughout the period of observation (Table II. Fig. 3). The superficial epithelium 43 hours after inoculation was cuboidal or low columnar, with domed 379
TABLE II. Mucosal Thickness (Am) of Cecum and Colon in Pairsof Control Dogs and Dogs at Times After Inoculation with Campylobacter jejuni Experimental Group 7 days 5 days 43 hours Control 350±45' 380+24 475±47 455±43 465±46 525±47 495±42 480±98 365±35 410±46 435±43 435±96 490±38 515±71 460±39 520±61 am - mean of 10 measurements ± SD
Organ Cecum Colon
superficial cell margins, loss of brush border and exfoliation of scattered cells evident in more severely affected regions (Fig. 4). Goblet cells were rarely present on
the surface. In the colon these changes were most severe on the surface of longitudinal folds. In the dog inoculated with CFJ1, erythrocytes and scattered neutro-
Fig. 2. Section of colonic mucosa from an uninoculated gnotobiotic control dog. Note the prevalence and distribution of goblet cells in glands and on the surface. H & E. X132.
Fig. 3. Cecal mucosa in a dog killed 43 hours after inoculation with C. jejuni. There is loss of goblet cells, attenuation of superficial epithelium, and elongation of glands. H & E. X132.
380
phils from underlying areas of congestion and hemorrhage were present in the epithelium and were moving into the lumen through microerosions (Fig. 4). The glands in dogs killed at this time contained, in many areas, fewer goblet cells than controls, and scattered glands were virtually devoid of mucous cells. Those remaining stained predominantly with Alcian blue. Glandular epithelium had more basophilic cytoplasm than in controls, and the proliferative compartment had expanded, with cells containing irregular large vesiculate nuclei and mitotic figures variably present up to half the depth of the gland. There was mild edema of the lamina propria and submucosa, associated with congestion and hemorrhage in the lamina propria of the tips of colonic folds in both dogs killed at 43 hours. Neutrophils were infiltrating the lamina propria between glands in the superficial half of the mucosa and were marginating in and migrating out of venules in the submucosa. Campylobacter were present in masses scattered over the surface, and in the lumen of the upper half of glands, with occasional individual organisms present in the base of glands. They did not appear to be within the cytoplasm of enterocytes, although occasional goblet cells which were extruding mucus seemed to have organisms within the mucus droplet. Scattered Staphylococci were present on the mucosal surface in most animals. In dogs killed five and seven days after inoculation, changes were still patchy and variable, but less acute. Superficial epithelium was attenuated, domed or low columnar and occasionally rounded up and exfoliating in some areas of cecum and colon in all four dogs, with more extensive and severe lesions in the cecum. Goblet cells were rarely seen on the surface, though their numbers had generally returned to normal in the upper portions of glands, and both acidic and basic mucopolysaccharide were present. The prolifera-
Fig. 4. Surface of cecal mucosa in a dog killed 43 hours after inoculation with C. jejuni. Superficial epithelium is attenuated and exfoliating, and a microerosion is present. There is a heavy neutrophil infiltrate in the lamina propria and neutrophils are effusing into the lumen through the eroded area. H & E. X330.
tive compartment continued to be phocytes were present in increased hypertrophic throughout this numbers between glands in the period with cells containing upper portion of the mucosa, and crowded irregular vesicular nuclei, neutrophils were infiltrating lambasophilic cytoplasm and many ina propria and submucosa in mitotic figures present in the basal some animals, though reduced in 25-60% of glands. Goblet cells were numbers in comparison with the sparser than normal in this portion 43 hour observation. Peyer's patches were well popuof glands. Scattered cystic glands containing mucus and necrotic lated by lymphocytes and were cells were present (Fig. 5). Lym- active in both control and Campy-
lobacter inoculated dogs. However, in the cecum and colon of Campylobacter infected animals, submucosal lymphoid follicles were much larger than the sparsely populated analogues in control animals, although mitotic activity did not appear high. The mesenteric lymph nodes of control dogs contained cortical follicles, and a moderate paracortical lymphocyte population, but mitotic activity was uncommon. In contrast, Campylobacter infected dogs had moderately hyperplastic lymph nodes, with primary and secondary follicles containing active germinal centres, and proliferating paracortical lymphocytes. Medullary trabeculae contained moderate numbers of lymphocytes, but plasma cells were rarely recognized at any time in lymph nodes or in gut mucosa. Examination by electron microscopy showed that the mucosal epithelium of the colon in puppies infected at 43 hours with either CFJ1 or CFJ14 contained low numbers of cells, which were sloughing into the lumen, into which the majority of goblet cells were discharging. Most epithelial cells appeared largely unaffected by the large numbers of Campylobacter in the lumen, although vacuolation of the endoplasmic reticulum was more marked in surface epithelial cells in comparison with those in control puppies. The microvilli of most epithelial cells were intact (Fig. 6) and no
Campylobacter were seen to invade intact epithelial cells, nor those few cells which were slough-
Fig. 5. Colonic mucosa in a dog killed five days after inoculation with C. jejuni. There is attenuation of surface epithelium, hyperplasia of cells in the basal half of glands, replacing goblet cells in that zone, and a cystic gland. H & E. X132.
ing into the lumen. In the colons of puppies infected for five and seven days, the only features of note were increased numbers of neutrophils, both within the epithelium and the lamina propria. Neutrophils did not contain Campylobacter, the goblet cells were not discharging, and epithelial cells seemed unaffected by the luminal Campylobacter. However, in a small number of neutrophils Staphylococci were visible. Campylobacter jejuni established itself in large numbers in 381
J.
Fig. 6. Electron micrograph of the surface epithelium in the colon of a dog inoculated with C. jejuni. Campylobacter present in the lumen in close association with normal microvillous border of a surface enterocyte. X19,600.
the intestine and could be detected readily in rectal swabs from 24 hours after infection. It established in greatest numbers in the colon (Table I) but was present in lower numbers throughout the remainder of the intestine. No. C. jejuni were present in heart blood but organisms were recovered in variable numbers from the majority of organs examined. No Campylobacter were isolated from control dogs. Staphylococci were also isolated in variable numbers from the body organs, the numbers and pattern of distribution in the tissues being similar to that shown for C. jejuni in Table I. Only one of the puppies infected with CFJ1 for seven days had an antibody titer, of 2, to C. jejuni; sera taken just before infection showed no antibodies.
DISCUSSION Our results demonstrate that high populations of C. jejuni of both human and canine origin are capable of causing a moderate superficial erosive colitis in gnotobiotic dogs, with signs of increasingly fluid feces and tenesmus at the height of the illness. Although the dogs had been bacteremic, 382
signs of systemic illness were limited to lassitude and mild inappetence and diarrhea was mild, in contrast to the disease in man in which abdominal pain and fever occur and blood may be present in the feces (4, 14, 15). Clinically, the experimental disease in dogs appears less severe than that in man, and this is consistent with some reports of the naturally occurring diarrhea in dogs associated with, but not proven to be due to, C. jejuni (1, 24). It is realized that small numbers of dogs were used in this study. The use of larger numbers of different breeds of dogs would have been desirable, but was ruled out by the cost of such studies. The pathological picture of the disease in dogs also appears milder than its human counterpart. Disease appeared limited to the cecum and colon, where the only significant gross finding was obvious congestion and edema of the mucosa along folds, and in the rectal area particularly. While this resembles the reports of some proctoscopic examinations in Campylobacter colitis of man (4, 20, 29), the biopsies illustrated by those authors appear more severely inflamed, with crypt abscessation in some cases. However, the gen-
eral pattern of loss of goblet cells, exfoliation of epithelium with microerosions and diapedesis of erythrocytes and neutrophils is similar in both species, and resembles mild lesions of swine dysentery in pigs (28) (a condition once associated with a vibrio, but now known to be caused by Treponema hyodysenteriae in concert with other anaerobes (10)). Since the gut flora in our dogs was restricted to Campylobacter and Staphylococcus, the role of Campylobacter as a primary pathogen seems clear, although the possibility of other bacteria exacerbating the condition in conventional dogs can not be overlooked. However, it has proven impossible to induce disease in conventional puppies in our laboratory (21). Staphylococci were isolated from both control and infected animals. It was not possible to conclude from the experiment that Staphylococci were not involved in the disease produced. Campylobacter jejuni was capable of inducing disease without invasion of the mucosa, implying that toxic factors released by organisms in close proximity to the mucosa may be causing the goblet cell discharge and epithelial exfoliation with erosion and acute inflammation observed within two days of inoculation. The colonic glands reacted by hypertrophy of the basal proliferative epithelial compartment (8, 16) which combined with the inflammatory edema, resulted in mucosal thickening. By five to seven days, epithelial production had apparently equilibrated with the increased rate of loss, and microerosions were not observed, although exfoliation and poor differentiation of surface epithelium were still evident. Crypt abscesses were not seen, but cystic crypts, probably the result of obstruction by edema and inflammatory infiltrate from the lamina propria, did occur. The nature of possible toxins is unknown and endotoxin involvement can not be eliminated on the present evidence. However, microscopic signs of systemic endotoxicosis were not seen in the
dogs killed at the height of disease, and thrombosis of the microvasculature was not found in inflamed colonic mucosa. Hyperplasia of lymphoid tissue in the submucosa and draining mesenteric lymph nodes implies a local immune reaction to infection, but plasma cells did not appear prominent in the inflammatory infiltrate at any time, and serum titers seven days after inoculation were low or nonexistent. While the disease and microscopic lesions in Campylobacter colitis of gnotobiotic dogs appear less severe than in man, it may prove a useful model for studies on the pathogenesis of this condition. It has not been established whether C. jejuni enteritis in man is primarily a small or large bowel infection; both parts of the intestine can be affected (4, 14). The difficulty experienced in producing Campylobacter enteritis in conventional dogs may relate to a failure of the organism to establish in large numbers in the intestine.
ACKNOWLEDGMENTS Dr. M.A. Karmali and Dr. M.J. Blaser kindly supplied isolates of C. jejuni. Dr. Don McKeown assisted with Caesarean section. The help of the staff of the Gnotobiotic Unit is appreciated. Ceciel Bruin-Mosch provided skilled technical assistance. This research was supported by a grant from the Ontario Ministry of Health (Number PR 824E), and in part by the Ontario Ministry of Agriculture and Food.
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