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Campylobacter hyointestinalis-associated enteritis in Moluccan rusa deer (Cervus timorensis subsp. Moluccensis)
(,‘AMPYLOBACTER
HYOINTESTZJVNAZ,IS-ASSOCIATED MOLUCCAN RUSA DEER (CERI’US TIMORENSIS SUBSP. MOLUCCENSIS)
ENTERITIS
B. D. Quernrland
Dvpartmen!
IN
HILL*,
qf Prima~v
and A. R. MACKENZIE?
R. J. THOMAS* *Animal Research Indus&s, Branch. Ipswch 4305.
Institute. Australia
lberongpil~v
410.5 and t Ciknnap
Srrm PS
INTRODUCTION
hyointestinalis has been isolated from pigs with lesions of proliferative ileitis (Gebhart, Ward, Chang and Kurtz, 1983; Gebhart, Edmonds, Ward, Kurtz and Brenner, 1985) and from the faeces of healthy calves (Walder, Sandstedt and Ursing, 1983; Myers, Firehammer, Border and Shoop, 1984). We report a Campylobacter r’yointestinalis-associated enteritis affecting Moluccan rusa deer on 2 farms in south-east Queensland. The enteritis manifested clinically as a persistent diarrhoea with chronic wasting. To our knowledge Campylobacter infection has not been described in deer. Campylobacter
MATERIALS
AND
METHODS
Herd Hislories The disease was studied on 2 farms (A and B), both of which reared Moluccan rusa and red deer (Ceruus elephus) in the same paddocks. On farm B, small numbers of fallow [Dama &ma) and chital (Axis axis) deer were also run in the same paddocks. The parent stock of rusa deer on farm A were captured on Prince of Wales Island in Torres Strait and were of mixed age and gender. Two years after introduction, signs of diarrhoea and wasting appeared and continued for 6 months. While farm A still had this problem, farm B established its rusa deer population by introducing clinically health? farm-bred rusa hinds from farm A and captured rusa deer from Prince of Wales Island. Diarrhoea on farm B commenced 12 months after introduction and continued for a further 11 months.
Pathology Six rusa hinds (2 pregnant) were submitted alive for necropsy. They were destroved and fresh tissues were fixed in 10 per cent buffered neutral formalin for lilqht microscopy. Formalin-fixed tissues were embedded in paraffin wax, sectioned 5 pm thick and stained with haematoxylin and eosin (HE). Sections of gastrointestinal tract were also stained by the Brown and Brenn (Humason, 1962) and Warthin-Starr\ ‘Armed Forces Institute of Pathology, 1960) methods for bacteria. Samples of formalin-fixed ileum and caecum were prepared for scanning electron microscopy. The fixed tissue was washed several times in phosphate buffer (0.5 M pH 7.2) and then post-fixed in 2 per cent osmium tetroxide (3 h) before being dehydrated in a graded series of ethanols. After dehydration, the tissue was take;, through a graded series of absolute ethanol and amyl acetate to 100 per cent am).1 acetate before being critical point dried. Specimens were mounted on stubs, sputte1 002 I
Y975/87/060687
+ 08$03.00/O
I(‘: 1987
Acadrmic
l’rcss
Limitcti
688
B.
D.
HILL
et.
al.
coated with platinum with a “Microvac” sputter coater and viewed in a Philips scanning electron microscope. Photographs were taken on Ilford FP4 film. Haematological, biochemical and parasitological examinations were performed standard methods.
505 by
Bacteriology Sampling Procedures. Faecal specimens were collected from the rectum. Heart blood, liver and spleen and contents ofjejunum, ileum, caecum and colon were sampled by sterile pipetting technique. Sections of small and large intestinal mucosa were gently washed free of ingesta with isotonic saline. The mucosa was scraped off with a scalpel blade and transferred to culture media by swab. Whole mesenteric lymph nodes were homogenized in nutrient broth with a stomacher (Colworth Laboratory Equipment) before culture. Isolation. Necropsy specimens were cultured aerobically at 37°C for 24 h on 5 per cent sheep blood agar (BA; Gibco), MacConkey agar (Oxoid) and brilliant green agar (Oxoid) with 0.008 per cent sodium sulphadiazine (before and after enrichment in Muller-Kauffmann tetrathionate broth (Oxoid)). Specimens from the last 2 necropsies were also cultured on Cumpylobacter agar (CA; Smeltzer, 1981) at 37°C for 48 h before and after enrichment in Cumpylobacter selective broth (CSB; Smeltzer, 1981). CSB contained the same antibiotics as CA and was incubated at 42°C for 72 h. CA plates were incubated in a microaerophilic atmosphere of 5 per cent O,, 10 per cent CO,, 20 per cent N, and 65 per cent H,. Faeces collected on the farms, from rusa deer with, (9 samples) and without, (15 samples) chronic diarrhoea were cultured on CA before and after enrichment in CSB. Samples of surface water and mud from one farm were cultured in an equal volume of double-strength CSB before plating on CA and wild bird droppings were enriched in CSB before plating on CA. Aerobically-incubated plates were examined visually for salmonellae and other enteropathogenic bacteria. Colonies suggestive of Campylobacter spp. on CA were subcultured to BA and incubated aerobically and in the microaerophilic atmosphere. The methods used to identify Campylobacter-like isolates were similar to those of Benjamin, Leaper, Owen and Skirrow (1983) except that antibiotic susceptibility tests were performed on unsupplemented yeast extract nutrient agar (YNA; 2.5 per cent Oxoid nutrient broth (NB) with 0.1 per cent yeast extract (Difco) and 2 per cent agar (Oxoid)). Susceptibility to 2,3,5-triphenyltetrazolium chloride (TTC) was determined on YNA only. Hippurate hydrolysis was evaluated by the method of Hwang and Ederer (1975) on 48 h BA cultures. Cellular morphology and motility were determined by dark ground microscopy on 24 h cultures in yeast nutrient broth (YNB; NB with O-1 per cent yeast extract (Difco) and 0.2 per cent agar (Oxoid)). Flagella arrangement, in a suspension in NB from a 24 h BA culture, was determined by electron microscopy after negative staining with 1 per cent phosphotungstic acid. The range of tests used to identify Campylobacter isolates and description of control strains, run in parallel with the unknown isolates, are given in Table 1. Soluble protein extracts were prepared from 24 h BA cultures of 3 isolates and the known Campylobacter strains listed in Table 1 by the method of Ferguson and Lambe by SDS-polyacrylamide gel electrophoresis (1984). These were examined (SDS-PAGE) by the method of Laemmli (1970)) with a 12 per cent resolving gel and 4 per cent stacking gel. RESULTS
Clinical
Findings
Of the 250 rusa
deer
on the two
farms,
diarrhoea
was seen only
in adults,
CAMPYLOBACTER
IN
RUSA
DEER
689
which ranged in age from one to 8 years. Twenty animals had died and approximately another 40, with persistent diarrhoea, eventually recovered. No new cases of diarrhoea have been observed on either farm. Examination of faeces from affected animals showed less than 50 helminth eggs per gram and no coccidial oocysts. Faeces were excessively fluid and in some cases contained flecks of blood. Haematological and biochemical examinations were made on blood samples from 11 rusa deer with persistent diarrhoea; 7 on farm cases and 4 of the cases submitted for necropsy. All showed neutrophilia, 10 with a left shift and 7 were hypoalbuminaemic.
Necropsied deer were 2 to 2.5 years of age and had been scouring for 1 to 2 months. All 6 cases were wasted, with fouling and loss of hair from the tail and perineal region. There was extensive serous atrophy of fat reserves. Towards the ileocaecal junction, the ileum was more distended and the mucosa more corrugated than normal. Surface mucus in the ileum of one animal contained numerous flecks of blood. There was an increased quantity of fluid in the caecum and colon of all cases. Microscopic lesions in the small intestine were confined to the ileum and the severity increased distally. There was a severe diffuse catarrhal enteritis characterized by stunting and fusion of villi and patchy erosion of superficial epithelium. While neutrophils predominated, increased numbers 01‘ lymphocytes and plasma cells were also present in the lamina propria and submucosa. Crypt epithelium was hyperplastic and often extended through gaps in the muscularis mucosae into the submucosa. The inflammator) reaction in caecum and colon was much less severe but focal erosion 01‘ superficial epithelium was still present. Brown and Brenn-stained sections of small and large intestine from all 6 cases showed moderate numbers of Gram-negative curved rods in close association with gland epithelium and invading the lamina propria where epithelial erosion had occurred. Thcsc bacteria were seen just as well in sections stained by the Warthin-Starry technique but could not be demonstrated to occupy the apical cytoplasm 01’ intestinal epithelial cells by either stain. Surface mucus adherent to the cpithelium was colonized by large numbers of these bacteria (Figs. 1 and 2 !. This was also,demonstrated by scanning electron microscopy of the caecum in one case where surface mucus was preserved (Fig. 3). Bacteriology
No salmonellae or other enteropathogenic bacteria were isolated on. the aerobically incubated media. Specimens cultured from the last 2 necropsy cases yielded small, watery beige-coloured colonies on CA after 48 h. These consisted of microaerophilic curved Gram-negative rods with darting motility which neither oxidized nor fermented glucose, were oxidase- and catalasepositive, grew well at 43°C and were resistant to nalidixic acid. These Cam#ylobacter isolates were cultured from the faeces, ileum, caecum, colon and
690
B.
D.
HILL
et. al.
Fig. 1. Large numbers of curved bacteria in surface mucus above the ileal mucosa. Brown and Brenn x 175. Fig. 2. A difkrent region of the ileum with curved bacteria in the surface mucus and entering the glands. Brown and Brenn x 175. Fig. 3. Sranning electron micrograph uf numerous curved bacteria on the surface of the caccal mucosa. x 2814.
mesenteric lymph nodes in both cases and from the spleen in one case. A Campylobacter isolate with the same characteristics was cultured from one farm-collected faecal specimen from a rusa deer with chronic diarrhoea. No Campylobacter or other enteropathogenic bacteria was cultured from the
CAMPYLOBACTER
IN
RUSA
DEER
69 I
remaining farm-collected faecal and environmental samples. A total of 23 specimens yielded these nalidixic acid-resistant thermophilic Camfiylobacter isolates, 7 by direct plating on CA but not after CSB enrichment, 4 after (ISB enrichment but not by direct CA plating and 12 by both methods. Three of the Campylobacter isolates (N3145, N7250 and N7751) were further tested. All had the same characteristics (Table I ). Very narrow zones of’ inhibition were present around 30 pg cephalothin discs on BA, but clear zones were evident on YNA. Electron microscopy revealed a single polar flagellum. SDS-PAGE did not indicate similarity to any of the known (;ampvlobac.terstrains tested. ‘The isolates were phenotypically similar to C. hyointe&ah, :i recent proposed species (Gebhart et al., 1983; Gebhart et nl., 1985). Our identification was confirmed by Dr. Connie Gebhart, University of Minnesota. St. Paul, MA USA.
N3145 N7250 N77Sl Catalase production Growth at: 25-c: 30.5”C 42°C: 43°C: 45.5”Ci Susceptibility to: Nalidixic acid (30 pg) ccphalothin (30 pg) rnetronidazole (5 pgi ‘l‘olrrancr to: ‘I‘TC I .O g per 1 ‘I-IT 0.4 g per 1 NaCl I .5 per cent NaCl 3-O per cent NaCl 3.5 per rent Glvcinr I.0 oer cent H,S producti& in TSI in YNB with h-ad awtatr strip in YNB plus FBP hippuratr hydrolysis a id liom gluros~ Nitrate rcdurtion Swarmirq on moist ~qa I
C. laridi?
+
+
+ %.I) + + + -
+” + + +
R s s
R
+
+ +
C. jejuni*
c. rolt*
6’.,jetus* sub. ,/e/u.r
+ +” + + +
+ + +”
2 s
R s R
+” +
-
t
-
f
-
+ + -
R s K
+ + +
+ + +
* Str‘lin
idrntification: C. landir, LNC’I’C 11352; C.jjuni, NCTC C:‘LI; C.&us sub. uenereafic, AR1 1980. t syrn bols: + x, wtakly positive; R, resistant; S, sensitive.
,/e&s, AR1
+ +
I 1168: (.‘. ~011, NC’I’C:
+ 11353; (.‘. /&us sub
DISCUSSION
A feature of the disease was light and electron-microscopical evidence of a strong association between Campylobacter-like bacteria and the intestinal surface mucus. A similar finding has been made with the human pathogen (:.,j@ni,
692
B. D. HILL
et. al.
which has been shown to be a mucus colonizer (Field, Underwood, Pope and Berry, 198 1; Lee, 1985). This evidence, together with bacteriological and other histopathological findings, support the C. hyointestinalis isolate as the possible aetiological agent of the diarrhoea. Electron-microscopical examination of faeces for a possible viral agent was not done. Considering the chronic nature of the diarrhoea and that the youngest animal affected was l-year-old, a viral aetiology was thought unlikely. Isolation of C. hyointestinalis from spleen and mesenteric lymph nodes demonstrated an ability to invade and survive beyond the intestinal mucosa. Immunofluorescence techniques (Chang, Kurtz, Ward and Gebhart, 1984) have been used to demonstrate occupation of enterocyte apical cytoplasm by C. hyointestinalis, in swine intestines with lesions of proliferative enteritis. With histological sections of deer intestine stained by Brown and Brenn and Warthin-Starry methods, we could not demonstrate intracellular bacteria. Transmission electron microscopy was not done. The pathogenesis of this diarrhoea is unclear but the stunting and fusion of villi, erosion of superficial epithelium and thickening of the lamina propria through inflammatory cell infiltration were likely to have contributed to malabsorption (Cheville, 1983). C. hyointestinalis was cultured from only one of 9 farm-collected faecal specimens from rusa deer with chronic diarrhoea. This indicates that either C. hyointestinalis was not present in these specimens or the organism was not detected by the isolation procedures used. The CA and CSB media were intended for isolation of C. jejuni. They contained cephalothin ( 15 mg per 1) which may have inhibited growth of C. hyointestinalis. Specimens for histopathological examination should be freshly fixed and care taken to preserve the surface mucus which contains large numbers of the bacteria. Examination of Gram-stained smears of ileal surface mucus may be useful. We are unable to suggest why the Moluccan rusa deer was the only breed, from the mixed populations on the farms, seen to develop this chronic wasting diarrhoea. The epidemiology of the disease is unknown and further studies are needed before control measures could be recommended. Rusa deer were acquired for the purpose of trying to reproduce this disease. Holding facilities available to us for keeping deer in isolation proved to be unsuitable and the experiment has been discontinued. of Campylobacter This study expands the host range for involvement hyointestinalis in enteric disease and is further evidence for mucus colonization as an important determinant of microbial pathogenicity. SUMMARY
A morphological and bacteriological study on a Campylobacter hyointestinalis-associated enteritis in adult Moluccan rusa deer is described. Necropsied deer were 2 to 2.5 years of age and had been scouring for 1 to 2 months. There was distension of the ileum and excessive corrugation of the mucosa. Microscopic lesions in the small intestine were confined to the ileum. Stunting and fusion of villi, patchy erosion of epithelium and a predominantly neutrophilic infiltrate were features. The inflammatory reaction in caecum and
CAMPYLOBACTER
IN
RUSA
DEER
693
colon was much less severe. Light and scanning electron-microscopical examination of small and large intestine showed large numbers of Gram-negative curved rods colonizing surface mucus and moder‘ate numbers in close association with gland epithelium. No salmonellae or other enteropathogenic bacteria were isolated on aerobically-incubated media. C. hyointestinalis was isolated from the faecea, ileum, caecum, colon and mesenteric lymph nodes from 2 cases and one t’arm-collected faecal sample. ACKNOWLEDGMENTS
\t:e thank H. C. Prior and D. H. Gowanlock fbr their assistance in preparing hpecirnens for scanning electron microscopy, Dr P. J. Walker and J. B. Molloy for assistance with PAGE and D. G. Rogers for preparation of media. We gratrfu.114 acknowledge the assistance given by Dr Gehhart in confirming our identification of (.. /~vointu.rlinalz.~.
REFERENCES
.\rmed Forces Institute of Pathology (1960). Manual of Histologic and Special Stain Technics. The Blakiston Division, McGraw-Hill Book Co. 2nd Edit. New York. Benjamin, J., Leaper, S., Owen, R. J. and Skirrow, M. B. ( 1983). Description of Cam~~lobacter laridis, a new species comprising the nalidixic acid resistant thermophilic Campylobacler (NARTC) group. C’urrenl Microbiology, 8, 231-238. (:haqg, I(., Kurtz, H. J., Ward, G. E. and Gebhart, C. J. (1984). Immunofluoresccnr demonstration of C’ampylobacter hyoinle.rtina1i.r and Cam/$obac/erium .spu/orum subsp. mucosa1i.s in swine intesiines with lesions of proliferatilre enteritis. .4meriran ,j’ourna/ oJ‘ CZterinar_y Research, 45, 703-7 10. (:he\ilie, N. 1:. (1983). Diseases of epithelium. In CPU Palholog)), 2nd Edit. ‘I‘he IO\V;I State University Press, Ames, Iowa, U.S.A. l:erguson,
694
B.
D.
HILL
et.
d.
Myers, L. L., Firehammer, B. D., Border, M. M. and of enteric pathogens in the faeces of healthy Veterinav Research, 45, 1546 1548. Smeltzer, T. I. (1981). Isolation of Cumpylobacterjejuni Veterinary Journal, 57, 5 1 1. Walder, M., Sandstedt, K. and Ursing, J. (1983). from human thermotolerant Campylobacter Microbiology, 9, 29 l-296. [Receivedfor publication,
314
Shoop, D. S. (1984). beef calves. American from poultry Phenotypic and animal
15th, 19861
Prevalence Journal
of
carcases. Australian characteristics of sources. Current
HYOINTESTZJVNAZ,IS-ASSOCIATED MOLUCCAN RUSA DEER (CERI’US TIMORENSIS SUBSP. MOLUCCENSIS)
ENTERITIS
B. D. Quernrland
Dvpartmen!
IN
HILL*,
qf Prima~v
and A. R. MACKENZIE?
R. J. THOMAS* *Animal Research Indus&s, Branch. Ipswch 4305.
Institute. Australia
lberongpil~v
410.5 and t Ciknnap
Srrm PS
INTRODUCTION
hyointestinalis has been isolated from pigs with lesions of proliferative ileitis (Gebhart, Ward, Chang and Kurtz, 1983; Gebhart, Edmonds, Ward, Kurtz and Brenner, 1985) and from the faeces of healthy calves (Walder, Sandstedt and Ursing, 1983; Myers, Firehammer, Border and Shoop, 1984). We report a Campylobacter r’yointestinalis-associated enteritis affecting Moluccan rusa deer on 2 farms in south-east Queensland. The enteritis manifested clinically as a persistent diarrhoea with chronic wasting. To our knowledge Campylobacter infection has not been described in deer. Campylobacter
MATERIALS
AND
METHODS
Herd Hislories The disease was studied on 2 farms (A and B), both of which reared Moluccan rusa and red deer (Ceruus elephus) in the same paddocks. On farm B, small numbers of fallow [Dama &ma) and chital (Axis axis) deer were also run in the same paddocks. The parent stock of rusa deer on farm A were captured on Prince of Wales Island in Torres Strait and were of mixed age and gender. Two years after introduction, signs of diarrhoea and wasting appeared and continued for 6 months. While farm A still had this problem, farm B established its rusa deer population by introducing clinically health? farm-bred rusa hinds from farm A and captured rusa deer from Prince of Wales Island. Diarrhoea on farm B commenced 12 months after introduction and continued for a further 11 months.
Pathology Six rusa hinds (2 pregnant) were submitted alive for necropsy. They were destroved and fresh tissues were fixed in 10 per cent buffered neutral formalin for lilqht microscopy. Formalin-fixed tissues were embedded in paraffin wax, sectioned 5 pm thick and stained with haematoxylin and eosin (HE). Sections of gastrointestinal tract were also stained by the Brown and Brenn (Humason, 1962) and Warthin-Starr\ ‘Armed Forces Institute of Pathology, 1960) methods for bacteria. Samples of formalin-fixed ileum and caecum were prepared for scanning electron microscopy. The fixed tissue was washed several times in phosphate buffer (0.5 M pH 7.2) and then post-fixed in 2 per cent osmium tetroxide (3 h) before being dehydrated in a graded series of ethanols. After dehydration, the tissue was take;, through a graded series of absolute ethanol and amyl acetate to 100 per cent am).1 acetate before being critical point dried. Specimens were mounted on stubs, sputte1 002 I
Y975/87/060687
+ 08$03.00/O
I(‘: 1987
Acadrmic
l’rcss
Limitcti
688
B.
D.
HILL
et.
al.
coated with platinum with a “Microvac” sputter coater and viewed in a Philips scanning electron microscope. Photographs were taken on Ilford FP4 film. Haematological, biochemical and parasitological examinations were performed standard methods.
505 by
Bacteriology Sampling Procedures. Faecal specimens were collected from the rectum. Heart blood, liver and spleen and contents ofjejunum, ileum, caecum and colon were sampled by sterile pipetting technique. Sections of small and large intestinal mucosa were gently washed free of ingesta with isotonic saline. The mucosa was scraped off with a scalpel blade and transferred to culture media by swab. Whole mesenteric lymph nodes were homogenized in nutrient broth with a stomacher (Colworth Laboratory Equipment) before culture. Isolation. Necropsy specimens were cultured aerobically at 37°C for 24 h on 5 per cent sheep blood agar (BA; Gibco), MacConkey agar (Oxoid) and brilliant green agar (Oxoid) with 0.008 per cent sodium sulphadiazine (before and after enrichment in Muller-Kauffmann tetrathionate broth (Oxoid)). Specimens from the last 2 necropsies were also cultured on Cumpylobacter agar (CA; Smeltzer, 1981) at 37°C for 48 h before and after enrichment in Cumpylobacter selective broth (CSB; Smeltzer, 1981). CSB contained the same antibiotics as CA and was incubated at 42°C for 72 h. CA plates were incubated in a microaerophilic atmosphere of 5 per cent O,, 10 per cent CO,, 20 per cent N, and 65 per cent H,. Faeces collected on the farms, from rusa deer with, (9 samples) and without, (15 samples) chronic diarrhoea were cultured on CA before and after enrichment in CSB. Samples of surface water and mud from one farm were cultured in an equal volume of double-strength CSB before plating on CA and wild bird droppings were enriched in CSB before plating on CA. Aerobically-incubated plates were examined visually for salmonellae and other enteropathogenic bacteria. Colonies suggestive of Campylobacter spp. on CA were subcultured to BA and incubated aerobically and in the microaerophilic atmosphere. The methods used to identify Campylobacter-like isolates were similar to those of Benjamin, Leaper, Owen and Skirrow (1983) except that antibiotic susceptibility tests were performed on unsupplemented yeast extract nutrient agar (YNA; 2.5 per cent Oxoid nutrient broth (NB) with 0.1 per cent yeast extract (Difco) and 2 per cent agar (Oxoid)). Susceptibility to 2,3,5-triphenyltetrazolium chloride (TTC) was determined on YNA only. Hippurate hydrolysis was evaluated by the method of Hwang and Ederer (1975) on 48 h BA cultures. Cellular morphology and motility were determined by dark ground microscopy on 24 h cultures in yeast nutrient broth (YNB; NB with O-1 per cent yeast extract (Difco) and 0.2 per cent agar (Oxoid)). Flagella arrangement, in a suspension in NB from a 24 h BA culture, was determined by electron microscopy after negative staining with 1 per cent phosphotungstic acid. The range of tests used to identify Campylobacter isolates and description of control strains, run in parallel with the unknown isolates, are given in Table 1. Soluble protein extracts were prepared from 24 h BA cultures of 3 isolates and the known Campylobacter strains listed in Table 1 by the method of Ferguson and Lambe by SDS-polyacrylamide gel electrophoresis (1984). These were examined (SDS-PAGE) by the method of Laemmli (1970)) with a 12 per cent resolving gel and 4 per cent stacking gel. RESULTS
Clinical
Findings
Of the 250 rusa
deer
on the two
farms,
diarrhoea
was seen only
in adults,
CAMPYLOBACTER
IN
RUSA
DEER
689
which ranged in age from one to 8 years. Twenty animals had died and approximately another 40, with persistent diarrhoea, eventually recovered. No new cases of diarrhoea have been observed on either farm. Examination of faeces from affected animals showed less than 50 helminth eggs per gram and no coccidial oocysts. Faeces were excessively fluid and in some cases contained flecks of blood. Haematological and biochemical examinations were made on blood samples from 11 rusa deer with persistent diarrhoea; 7 on farm cases and 4 of the cases submitted for necropsy. All showed neutrophilia, 10 with a left shift and 7 were hypoalbuminaemic.
Necropsied deer were 2 to 2.5 years of age and had been scouring for 1 to 2 months. All 6 cases were wasted, with fouling and loss of hair from the tail and perineal region. There was extensive serous atrophy of fat reserves. Towards the ileocaecal junction, the ileum was more distended and the mucosa more corrugated than normal. Surface mucus in the ileum of one animal contained numerous flecks of blood. There was an increased quantity of fluid in the caecum and colon of all cases. Microscopic lesions in the small intestine were confined to the ileum and the severity increased distally. There was a severe diffuse catarrhal enteritis characterized by stunting and fusion of villi and patchy erosion of superficial epithelium. While neutrophils predominated, increased numbers 01‘ lymphocytes and plasma cells were also present in the lamina propria and submucosa. Crypt epithelium was hyperplastic and often extended through gaps in the muscularis mucosae into the submucosa. The inflammator) reaction in caecum and colon was much less severe but focal erosion 01‘ superficial epithelium was still present. Brown and Brenn-stained sections of small and large intestine from all 6 cases showed moderate numbers of Gram-negative curved rods in close association with gland epithelium and invading the lamina propria where epithelial erosion had occurred. Thcsc bacteria were seen just as well in sections stained by the Warthin-Starry technique but could not be demonstrated to occupy the apical cytoplasm 01’ intestinal epithelial cells by either stain. Surface mucus adherent to the cpithelium was colonized by large numbers of these bacteria (Figs. 1 and 2 !. This was also,demonstrated by scanning electron microscopy of the caecum in one case where surface mucus was preserved (Fig. 3). Bacteriology
No salmonellae or other enteropathogenic bacteria were isolated on. the aerobically incubated media. Specimens cultured from the last 2 necropsy cases yielded small, watery beige-coloured colonies on CA after 48 h. These consisted of microaerophilic curved Gram-negative rods with darting motility which neither oxidized nor fermented glucose, were oxidase- and catalasepositive, grew well at 43°C and were resistant to nalidixic acid. These Cam#ylobacter isolates were cultured from the faeces, ileum, caecum, colon and
690
B.
D.
HILL
et. al.
Fig. 1. Large numbers of curved bacteria in surface mucus above the ileal mucosa. Brown and Brenn x 175. Fig. 2. A difkrent region of the ileum with curved bacteria in the surface mucus and entering the glands. Brown and Brenn x 175. Fig. 3. Sranning electron micrograph uf numerous curved bacteria on the surface of the caccal mucosa. x 2814.
mesenteric lymph nodes in both cases and from the spleen in one case. A Campylobacter isolate with the same characteristics was cultured from one farm-collected faecal specimen from a rusa deer with chronic diarrhoea. No Campylobacter or other enteropathogenic bacteria was cultured from the
CAMPYLOBACTER
IN
RUSA
DEER
69 I
remaining farm-collected faecal and environmental samples. A total of 23 specimens yielded these nalidixic acid-resistant thermophilic Camfiylobacter isolates, 7 by direct plating on CA but not after CSB enrichment, 4 after (ISB enrichment but not by direct CA plating and 12 by both methods. Three of the Campylobacter isolates (N3145, N7250 and N7751) were further tested. All had the same characteristics (Table I ). Very narrow zones of’ inhibition were present around 30 pg cephalothin discs on BA, but clear zones were evident on YNA. Electron microscopy revealed a single polar flagellum. SDS-PAGE did not indicate similarity to any of the known (;ampvlobac.terstrains tested. ‘The isolates were phenotypically similar to C. hyointe&ah, :i recent proposed species (Gebhart et al., 1983; Gebhart et nl., 1985). Our identification was confirmed by Dr. Connie Gebhart, University of Minnesota. St. Paul, MA USA.
N3145 N7250 N77Sl Catalase production Growth at: 25-c: 30.5”C 42°C: 43°C: 45.5”Ci Susceptibility to: Nalidixic acid (30 pg) ccphalothin (30 pg) rnetronidazole (5 pgi ‘l‘olrrancr to: ‘I‘TC I .O g per 1 ‘I-IT 0.4 g per 1 NaCl I .5 per cent NaCl 3-O per cent NaCl 3.5 per rent Glvcinr I.0 oer cent H,S producti& in TSI in YNB with h-ad awtatr strip in YNB plus FBP hippuratr hydrolysis a id liom gluros~ Nitrate rcdurtion Swarmirq on moist ~qa I
C. laridi?
+
+
+ %.I) + + + -
+” + + +
R s s
R
+
+ +
C. jejuni*
c. rolt*
6’.,jetus* sub. ,/e/u.r
+ +” + + +
+ + +”
2 s
R s R
+” +
-
t
-
f
-
+ + -
R s K
+ + +
+ + +
* Str‘lin
idrntification: C. landir, LNC’I’C 11352; C.jjuni, NCTC C:‘LI; C.&us sub. uenereafic, AR1 1980. t syrn bols: + x, wtakly positive; R, resistant; S, sensitive.
,/e&s, AR1
+ +
I 1168: (.‘. ~011, NC’I’C:
+ 11353; (.‘. /&us sub
DISCUSSION
A feature of the disease was light and electron-microscopical evidence of a strong association between Campylobacter-like bacteria and the intestinal surface mucus. A similar finding has been made with the human pathogen (:.,j@ni,
692
B. D. HILL
et. al.
which has been shown to be a mucus colonizer (Field, Underwood, Pope and Berry, 198 1; Lee, 1985). This evidence, together with bacteriological and other histopathological findings, support the C. hyointestinalis isolate as the possible aetiological agent of the diarrhoea. Electron-microscopical examination of faeces for a possible viral agent was not done. Considering the chronic nature of the diarrhoea and that the youngest animal affected was l-year-old, a viral aetiology was thought unlikely. Isolation of C. hyointestinalis from spleen and mesenteric lymph nodes demonstrated an ability to invade and survive beyond the intestinal mucosa. Immunofluorescence techniques (Chang, Kurtz, Ward and Gebhart, 1984) have been used to demonstrate occupation of enterocyte apical cytoplasm by C. hyointestinalis, in swine intestines with lesions of proliferative enteritis. With histological sections of deer intestine stained by Brown and Brenn and Warthin-Starry methods, we could not demonstrate intracellular bacteria. Transmission electron microscopy was not done. The pathogenesis of this diarrhoea is unclear but the stunting and fusion of villi, erosion of superficial epithelium and thickening of the lamina propria through inflammatory cell infiltration were likely to have contributed to malabsorption (Cheville, 1983). C. hyointestinalis was cultured from only one of 9 farm-collected faecal specimens from rusa deer with chronic diarrhoea. This indicates that either C. hyointestinalis was not present in these specimens or the organism was not detected by the isolation procedures used. The CA and CSB media were intended for isolation of C. jejuni. They contained cephalothin ( 15 mg per 1) which may have inhibited growth of C. hyointestinalis. Specimens for histopathological examination should be freshly fixed and care taken to preserve the surface mucus which contains large numbers of the bacteria. Examination of Gram-stained smears of ileal surface mucus may be useful. We are unable to suggest why the Moluccan rusa deer was the only breed, from the mixed populations on the farms, seen to develop this chronic wasting diarrhoea. The epidemiology of the disease is unknown and further studies are needed before control measures could be recommended. Rusa deer were acquired for the purpose of trying to reproduce this disease. Holding facilities available to us for keeping deer in isolation proved to be unsuitable and the experiment has been discontinued. of Campylobacter This study expands the host range for involvement hyointestinalis in enteric disease and is further evidence for mucus colonization as an important determinant of microbial pathogenicity. SUMMARY
A morphological and bacteriological study on a Campylobacter hyointestinalis-associated enteritis in adult Moluccan rusa deer is described. Necropsied deer were 2 to 2.5 years of age and had been scouring for 1 to 2 months. There was distension of the ileum and excessive corrugation of the mucosa. Microscopic lesions in the small intestine were confined to the ileum. Stunting and fusion of villi, patchy erosion of epithelium and a predominantly neutrophilic infiltrate were features. The inflammatory reaction in caecum and
CAMPYLOBACTER
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colon was much less severe. Light and scanning electron-microscopical examination of small and large intestine showed large numbers of Gram-negative curved rods colonizing surface mucus and moder‘ate numbers in close association with gland epithelium. No salmonellae or other enteropathogenic bacteria were isolated on aerobically-incubated media. C. hyointestinalis was isolated from the faecea, ileum, caecum, colon and mesenteric lymph nodes from 2 cases and one t’arm-collected faecal sample. ACKNOWLEDGMENTS
\t:e thank H. C. Prior and D. H. Gowanlock fbr their assistance in preparing hpecirnens for scanning electron microscopy, Dr P. J. Walker and J. B. Molloy for assistance with PAGE and D. G. Rogers for preparation of media. We gratrfu.114 acknowledge the assistance given by Dr Gehhart in confirming our identification of (.. /~vointu.rlinalz.~.
REFERENCES
.\rmed Forces Institute of Pathology (1960). Manual of Histologic and Special Stain Technics. The Blakiston Division, McGraw-Hill Book Co. 2nd Edit. New York. Benjamin, J., Leaper, S., Owen, R. J. and Skirrow, M. B. ( 1983). Description of Cam~~lobacter laridis, a new species comprising the nalidixic acid resistant thermophilic Campylobacler (NARTC) group. C’urrenl Microbiology, 8, 231-238. (:haqg, I(., Kurtz, H. J., Ward, G. E. and Gebhart, C. J. (1984). Immunofluoresccnr demonstration of C’ampylobacter hyoinle.rtina1i.r and Cam/$obac/erium .spu/orum subsp. mucosa1i.s in swine intesiines with lesions of proliferatilre enteritis. .4meriran ,j’ourna/ oJ‘ CZterinar_y Research, 45, 703-7 10. (:he\ilie, N. 1:. (1983). Diseases of epithelium. In CPU Palholog)), 2nd Edit. ‘I‘he IO\V;I State University Press, Ames, Iowa, U.S.A. l:erguson,
694
B.
D.
HILL
et.
d.
Myers, L. L., Firehammer, B. D., Border, M. M. and of enteric pathogens in the faeces of healthy Veterinav Research, 45, 1546 1548. Smeltzer, T. I. (1981). Isolation of Cumpylobacterjejuni Veterinary Journal, 57, 5 1 1. Walder, M., Sandstedt, K. and Ursing, J. (1983). from human thermotolerant Campylobacter Microbiology, 9, 29 l-296. [Receivedfor publication,
314
Shoop, D. S. (1984). beef calves. American from poultry Phenotypic and animal
15th, 19861
Prevalence Journal
of
carcases. Australian characteristics of sources. Current