Arcobacter (Campylobacter) - Infection and Immunity - American ...

INFECTION AND IMMUNITY, May 1993, p. 2220-2223 0019-9567/93/052220-04$02.00/0

Vol.

61, No. 5

Copyright X 1993, American Society for Microbiology

Arcobacter (Campylobacter) butzleri-Associated Diarrheal Illness in a Nonhuman Primate Population KAREN F.

ANDERSON,"*

JULIA A.

KIEHLBAUCH,2t

DANIEL C. ANDERSON,'

HAROLD M. McCLURE,1 AND I. KAYE WACHSMUTH2 Division of Pathobiology and Immunobiology, Yerkes Primate Research Center, Emory University, Atlanta, Georgia 30322,1 and Enteric Diseases Laboratory Section, Division of Bacterial and Mycotic Diseases, National Centers for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 29 September 1992/Accepted

5

February 1993

After DNA hybridization identified an isolate from an ill rhesus macaque (Macaca mulatta) as Arcobacter (Campylobacter) butzieri, we initiated a study to determine whether A. butzleri was associated with diarrheal disease in nonhuman primates at the Yerkes Primate Research Center. By using Campy-CVA medium incubated at 35'C, 15 A. butzleri isolates were obtained from 14 macaques; 7 macaques were coinfected with Campylobacter coli and Campylobacterjejuni. A. butzleri was not isolated from normal feces, despite the fact that feces from 76 macaques were cultured at necropsy. Histologic evaluation of colonic specimens from three macaques from which A. butzleri had been isolated showed mild to moderately severe chronic, active colitis. Ribotype analysis of the 15 A. butzlri isolates revealed nine different strains; these data suggest that A. butzleri may be endemic in this primate population and that a point source of infection is unlikely. This is the first report of the presence of A. butzkeri in juvenile and adult macaques with diarrhea, and it may present an opportunity to study the pathogenesis of this organism, which appears to be associated with persistent diarrhea in humans. Recent investigation of a group of isolates from human and animal sources, putatively thought to be Campylobacter cryaerophila on the basis of aerotolerance, revealed three DNA hybridization groups (7). DNA hybridization group 1A, C. cryaerophila, consisted primarily of bovine and porcine strains isolated from tissue specimens, while hybridization group 1B contained several strains of both human and animal origins. DNA hybridization group 2 strains, Campylobacter butzleri, were primarily from humans and nonhuman primates with diarrhea (7). Recently, other investigators have proposed that C. cryaerophila and C. butzleri be moved to a new genus, Arcobacter (23, 25). Nine nonhuman primate isolates included in the hybridization study (7) were identified as Arcobacter (Campylobacter) butzlen; one was recovered in 1986 from a 2-month-old rhesus macaque (Macaca mulatta) with diarrheal illness, and eight isolates were part of the study reported here. To identify further isolates of A. butzlen, 532 diarrheal specimens (diarrhea defined as increased volume or increased frequency) from 222 nonhuman primates housed either in social groups in large corrals or in individual suspended stainless steel caging were submitted for laboratory examination at Yerkes Primate Research Center over an 8-month period. (The Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care.) In addition, 76 macaque colonic specimens were routinely cultured for enteric pathogens as part of the necropsy protocol. All specimens were cultured in parallel at 42'C on Campy-BAP, which contains vancomycin, polymyxin B, trimethoprim, cycloheximide, and cephalothin (BBL, Becton Dickinson Microbiological Systems, Cockeysville, Md.) (2), and at 35'C on Campy-CVA, which contains cefoperazone, vancomycin, and amphotericin B

(BBL) (14). All plates were incubated in a microaerobic atmosphere produced by either a CampyPak Plus system envelope (BBL) or a Campy Pouch system (BBL) for 72 h. Specimens were also cultured on MacConkey, salmonellashigella, Hektoen, and cefsulodin-Irgasan-novobiocin (CIN) agars (all from BBL) for Salmonella, Shigella, and Yersinia spp. by standard microbiologic techniques. We retrospectively reviewed clinical records and examined available biopsy or necropsy tissues of animals from which A. butzleri was isolated, using hematoxylin and eosin and modified Steiner silver stains (20, 21). In addition, A. butzlen isolates were characterized by phenotype (1) and by ribotype by using PvuII and ClaI for species identification and epidemiologic purposes, as previously described (8). Isolates were classified as A. butzleri if they demonstrated common bands at 3.0, 7.2, 12.0, and 15.0 kb in PvuII digests (8). Patterns were determined following visual inspection; different strains of A. butzlen were identified by differences in patterns in genes coding for rRNA. During the 8-month study, we identified A. butzleri infections in 14 of 222 diarrheic animals (Table 1); only one animal yielded A. butzleri isolates on more than one occasion. One animal with diarrhea yielded A. butzleri isolates only at necropsy, despite having two cultures prior to death. Seven of the 14 animals with A. butzlen infections were concomitantly infected with Campylobacter coli and Campylobacter jejuni, but no other potential enteric pathogens were identified. At the time of A. butzlen isolation, 3 animals (OPE342, RKg, and RWk) were housed in outdoor corrals and 11 were housed in individual cages; however, 6 (PVj, RDb-1, ROo, HLb, RFp, and RSn-2) had been transferred from outdoor housing within the preceding 3 months because of diarrhea. Three of these animals (RDb-1, HLb, and RFp) were in individual cages in the same room at the time A. butzlen was isolated (Table 1). In contrast, during the 8-month study period, colonic specimens from 76 macaques (57 rhesus, 7 pig-tailed, and 12 stump-tailed) .6 months old were cultured. These speci-

* Corresponding author. t Present address: Department of Pathology, Medical College of Wisconsin, University of Wisconsin, Milwaukee, WI 53226.

2220

VOL. 61, 1993

NOTES

2221

TABLE 1. Summary of study of diarrheic macaques with A. butzleri infection Macaque no.

Species

Age

Sex

RSn-2 RUk-2 OPE221 RDb-1 ROo RZt CF40 OPE342 RWk RKg RFp PVj HLb MF403

Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Rhesus Pig-tailed Stump-tailed Cynomolgus

7 mo 10 mo 5 yr 6 yr 6 yr 7 yr 7 yr 11 yr 14 yr 17 yr 17 yr 4 yr 8 yr 6 yr

F F F F F M F F F M F F M F

a Includes all enteric pathogens, bacterial and parasitic.

Other enteric pathogen isolated0

Location at time of positive culture"

C. jejuni C. coli C. coli

C. coli C coli

SF-1-242 SF-2-10 HF-5 HF-3 SF-2-4 SF-3-105 Outdoors Outdoors Outdoors HF-5 HF-3

TABLE 2. Summary of clinical and necropsy culture results No. of positive specimens from:

Organism

A. butzlen

C. jejuni C coli Campylobacter fetus subsp. fetus Shigella fiexneri Salmonella typhimunum Yersinia spp.c Total positive cultures

Animals with

Macaques mo old

diarrhea0

26 (necropsy)b

14 85 212 7 37 0 9

1 5 10 3 3 2 5

268

25

a A total of 532 specimens from 222 animals were tested. The number of animals with multiple Campylobacter spp. was 22. b A total of 76 specimens from 76 animals were tested (without regard to diarrheal status). The number of animals with multiple Campylobacter spp.

was 4. c Includes 6 Y enterocolitica, 6 Y. pseudotuberculosis, and 2 Y senii isolates and 1 Y. kristensenii isolate.

frederik-

swabc swabc swab swab swab

Colicd Fecal Fecal Rectal swab

HF-5 SF-2-5

bHF, hospital facility (numbers indicate room numbers); SF, standard indoor facility (first and second numbers indicate respectively). c Histology done on biopsy colonic specimen. d Culture and histologic specimens obtained at necropsy.

mens were obtained from outdoor-housed (45 macaques) and individually caged (31 macaques) animals (54% females) submitted for routine postmortem examination, regardless of diarrheal illness at the time of death. Only one additional A. butzleri infection (RFp; Table 1) was found, and records indicated a diarrheal illness at death. Results from clinical and necropsy cultures are summarized in Table 2. A. butzleri was isolated from three animals at the time of biopsy or necropsy. For two of them, A. butzleri was the only potential enteric pathogen isolated; the third animal had a concomitant C. jejuni infection (Table 1). Histologic examination revealed chronic active colitis in all three tissues examined (Fig. 1). Silver stains revealed Campylobactershaped organisms in all three cases. Of the 76 macaques submitted for necropsy, 73 had colonic and small-intestinal tissues suitable for histologic evaluation; 41% were histologically normal, and 23% had chronic active colitis (Table 3). The prevalence of chronic active colitis in A. butzleni culture-negative animals (17 of 72) was significantly lower than

Fecal Fecal Fecal Rectal Rectal Rectal Fecal Rectal Rectal Fecal

HF-5 C. jejuni C. coli

Type of specimen

building

and room numbers,

the prevalence of similar lesions in A. butzleri culturepositive animals (3 of 3) (by a two-tailed Fisher's exact test, P = 0.017). The ribotypes of the 15 A. butzleri isolates are shown in Fig. 2. This analysis identified nine strains (or patterns) among the 15 isolates; eight different strains were isolated from rhesus macaques, and one additional strain was isolated from a stump-tailed macaque (Macaca arctoides). The most common strain, ribotype A, was isolated from three macaque species: one cynomolgus (Macaca fasciculanis), one pig-tailed (Macaca nemestnina), and three rhesus (M. mulatta) macaques; each species was housed in a separate facility. Two pattern A isolates were from rhesus monkeys housed in individual cages in the same room at the time of isolation. A second isolate, obtained from the pig-tailed macaque (PVj) 3 weeks later, was the same strain (ribotype A) as the first isolate. Ribotype data confirmed a common 3.0-kb band in PvuII digests in all A. butzleri isolates; this band appears to be specific to A. butzleri and is not found in other closely related species (8). Although high rates of Campylobacter isolation are common in laboratory-housed nonhuman primate populations (4, 6, 10, 12, 15, 17-19), little is known about the potential pathogenic role of campylobacter-like organisms other than C. jejuni and C. coli in nonhuman primate disease. Campylobacter species and related organisms are increasingly recognized as major causes of human illness. To the best of our knowledge, this is the first report of A. butzleri, a newly described aerotolerant species, in juvenile and adult nonhuman primates. Other investigators have described organisms that were later shown to be A. butzleri strains from asymptomatic infant pig-tailed macaques (3, 16). To date, we have isolated A. butzleri only from diarrheic macaques >6 months old. The low incidence of A. butzleri (6% of animal specimens cultured) prevented us from conducting a true case-control study. However, we did not isolate A. butzleri from animals with normal colons, despite the routine screening of animals for enteric pathogens at death regardless of the presence of diarrheal illness. The pathologic features reported here were nonspecific, since the histologic lesions in animals with A. butzleri infection in this report were virtually identical to those in

2222

INFECT. IMMUN.

NOTES

FIG. 1. High magnification of hematoxylin and eosin stain of a colonic specimen from a rhesus monkey, showing acute cryptitis with neutrophils within crypt lumens and extending into the lamina propria. The lamina propria is generally hypercellular, predominantly plasmacytic or lymphocytic, with few histiocytes. Magnification, x450; bar, 20 p.m.

humans with Campylobacter colitis (5, 11) and in monkeys with experimental C. jejuni infection (15). The lesions have features of both acute and chronic inflammation; thus, these lesions may be interpreted as consistent with those produced

Kb

TABLE 3. Comparison of animals tested clinically and at necropsy Macaque species and age

Rhesus 6mo-lyr 1-5 yr 6-lOyr 11-15 yr 16-20 yr >20 yr

No. of clinical cases

No. of animals tested at necropsy

Normal

With other

inflammation

lesionsa

0 2 4 4

2 1 4 2 2 0

1 4 8 3 6 5

0

1 6 3 2 2 1

Total

11

27

13b

15

Pig-tailed

1

1

4

2

Stump-tailed

1

2

1

8c

13

30

18

25

Total

1 2 3 4

23.1-

With chronic active

3b

by other inflammatory causes of colitis, including infectious agents (5, 15). Ribotype data identified a widespread distribution of one strain thoughout the colony (and in several macaque species)

a Lesions ranged from mild inflammation to severe amyloidosis. b Includes one rhesus macaque with A. butzleri infection. c Natural simian immunodeficiency virus infection and mycobacteriosis.

5 6 7 8 9 10 11 121314151617

jX

12.2- 3

10.1-

Wv _3w r=ws -0b " *Ewgete *

3.01-

.0 .0

.b

O* iL 94

FIG. 2. Patterns of ribosomal DNA from aerotolerant Campylobacter isolates described in this report. Lane 1, A. butzleri type strain (D2686 = ATCC 49616). Lanes 2 to 16, isolates from 14 macaques; lane 2, MF 403, pattern A; lane 3, OPE342, pattern B; lane 4, RKg, pattern C; lane 5, RZt, pattern D; lane 6, PVj (first isolate), pattern A; lane 7, PVj (second isolate), pattern A; lane 8, RWk, pattern E; lane 9, RDb-1, pattern A; lane 10, ROo, pattern B; lane 11, HLb, pattern F; lane 12, RFp, pattern A; lane 13, RUk-2, pattern G; lane 14, RSn-2, pattern H; lane 15, OPE221, pattern A; lane 16, CF40, pattern I. Lane 17, C cryaerophila DNA hybridization group 1B type strain (D2610 = ATCC 49615).

VOL. 61, 1993

in addition to seven other strains isolated from rhesus monkeys. This suggests that there may be several foci of infection rather than a single source. A. butzleni appears to be endemic in this primate population, with a low incidence of infection or colonization. The macaques may have been infected by consumption of contaminated standing water or food contaminated with fecal material in outdoor corrals. Preliminary investigations of human illness associated with A. butzleri indicate an association between isolation of A. butzleri and exposure to surface water (9). In addition, bacteria consistent with A. butzleri were isolated from water during a study of a French urban public water supply (13). A. butzleri has been associated with acute diarrheal illness (22), recurrent abdominal cramps (24), and persistent diarrhea in humans (7, 9). Our data indicate that A. butzlen is associated with diarrheal illness in nonhuman primates and should be evaluated as a potential enteric pathogen. Welldesigned case-control studies are difficult to conduct with nonhuman primates because of the low incidence of A. butzleri infection and the inherent difficulty in obtaining accurate diarrhea histories in a group setting. However, further surveillance and feeding experiments will better determine the pathogenesis of A. butzlen in nonhuman primates and may lead to a better understanding of the role of this organism in human diarrheal illness. This work at Yerkes Primate Research Center was supported in part by Public Health Service grant RRO0165 from the National Institutes of Health and was completed while J.A.K. held a National Research Council-Centers for Disease Control research associate-

ship. We also acknowledge the assistance of Annelise Green in the staining of histology sections. REFERENCES 1. Barrett, T. J., C. M. Patton, and G. K. Morris. 1988. Differentiation of Campylobacter species using phenotypic characterization. Lab. Med. 19:96-102. 2. Blaser, M. J., D. Berkowitz, F. M. Laforce, J. Cravens, L. B. Reller, and W.-L. L. Wang. 1979. Campylobacter enteritis: clinical and epidemiological features. Ann. Intern. Med. 91:179185. 3. Bronsdon, M. 1985. Unusual Campylobacter species isolated from stool of non-human primates, abstr. C130, p. 321. Abstr. Annu. Meet. Am. Soc. Microbiol. 1985. 4. Bryant, J. L., H. F. Stills, R. H. Lentsch, and C. C. Middleton. 1983. Campylobacter jejuni isolated from Patas monkeys with diarrhea. Lab. Anim. Sci. 33:303-305. 5. Coffin, C. M., P. L'Heureaux, and L. P. Dehner. 1982. Campylobacter-associated enterocolitis in childhood. Am. J. Clin. Pathol. 78:117-123. 6. Fox, J. G. 1982. Campylobacteriosis-a "new" disease in laboratory animals. Lab. Anim. Sci. 32:625-637. 7. Kiehlbauch, J. A., D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth. 1991. Campylobacter butzleri sp. nov. isolated from humans and animals with diarrheal illness. J. Clin. Microbiol. 29:376-385. 8. Kiehlbauch, J. A., B. D. Plikaytis, B. Swaminathan, D. N. Cameron, and L. K. Wachsmuth. 1991. Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species. J. Clin. Microbiol. 29:1670-1676.

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