Persistent, recurring diarrhea in a colony of orangutans (Pongo pygmaeus) caused by multiple strains of Campylobacter spp.

Acta Tropica, 57(1994)1 l0 © 1994 Elsevier Science B.V. All rights reserved 0001-706X/94/$07.00

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ACTROP 00370

Persistent, recurring diarrhea in a colony of orangutans (Pongopygmaeus) caused by multiple strains of Campylobacterspp. G. Pazzaglia a'*, S. Widjaja a, D. Soebekti a, P. Tjaniadi a, L. Simanjuntak b, M. Lesmana", G. Jennings" aU.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia, bRagunan Zoo, Jakarta, Indonesia Received 4 October 1993; accepted 27 December 1993

A colony of 10 orangutans (Pongo pygmaeus) experienced persistent, recurring diarrhea caused by multiple infections with Campylobacter jejuni and C. coli. Infections appeared to have occurred through several mechanisms, including fecal-oral transmission between orangutans, and possibly transmission by houseflies contaminated with the organisms from nearby chicken feces. Among the 14 fecal and environmental C. jejuni isolates, 4 different antibiotic susceptibility profiles were detected; there were also 4 different profiles among the 8 isolates of C. coli. In 5 orangutans, there were back-to-back infections by different strains of C. jejuni, suggesting that a single C. jejuni infection may not confer protective immunity against heterologous strains circulating in the same vicinity. Transmission was effectively interrupted by environmental modifications and a 7-day course of oral erythromycin. Key words: Campylobacter; Diarrhea; Orangutan; Pongopygmaeus; Antibiotic

Introduction

Since the development of the first Campylobacter-selective medium by Skirrow (1977), the worldwide significance of Campylobacter spp. as an important cause of human diarrhea, and as a frequent isolate from the environment, foods, and dozens of animal species, have become apparent (Blaser et al., 1983; Fox, 1982; Harris et al., 1986; Leuchtefeld et al., 1981 ). There is a broad spectrum of clinical syndromes associated with Campylobacter spp. infection in humans, ranging from frank bloody diarrhea and fever to watery diarrhea to asymptomatic excretion (Blaser and Reller, 1981; Glass et al., 1983; Pazzaglia et al., 1991). In the United States and other industrialized countries, campylobacteriosis occurs sporadically; contaminated foods of animal origin, in particular raw milk and poultry, are most frequently the sources of infection for individual cases, as well as periodic outbreaks. In many tropical developing countries, human Campylobacter infections are hyperendemic and the organisms are frequently isolated from *Corresponding author. Present address." Naval Medical Research Institute, NNMC, 8901 Wisconsin Ave., Bethesda, MD 20889-5607, USA. For reprints." Publications Office, U.S. NAMRU-2 Box 3, Unit 8132, APO AP 96520 8132 USA. SSDI 0 0 0 1 - 7 0 6 X ( 9 4 ) 0 0 0 0 4 - K

household environments, foods, and water sources (Georges-Courbot et al., 1987; Pazzaglia et al., 1993; Rajan and Mathan, 1982). Because of its wide distribution in the environment, levels of asymptomatic carriage are high, often reaching 30 to 40% in young children (Pazzaglia et al., 1991; Blaser et al., 1980). Animal surveys have isolated Campylobacterspp. from feces of many species of wild, domestic, and laboratory animals (Fox, 1982; Ackerman et al., 1982; Butzler, 1984). Although frequently isolated, the importance of Campylobacter spp. as a cause of diarrhea in nonprimate species is unclear; the organisms most commonly colonize the bowel as commensals, resulting in no discernible ill effects to the host (Fox, 1982). The role of Campylobacter spp. as a cause of diarrhea in monkeys and other nonhuman primates appears to be similar to that shown for human infections. The organisms have been isolated from apparently healthy monkeys (Fox, 1982), but there have been numerous reports of Campylobacter strains being the cause of diarrhea in several New World and Old World species of monkeys (Bryant et al., 1983; Morton et al., 1981; Tribe and Frank, 1980). Evidence suggests the organisms are probably not commensals (or natural pathogens) of simians in the wild, and that captured primates may become infected during their early stages of captivity or quarantine in tropical developing countries (Morton et al., 1983). Campylobacterassociated diarrhea is an important clinical problem in institutions housing primates for research (Hird et al., 1984). There are few reports of campylobacteriosis in apes; Campylobacter spp. were reported as a possible cause of hemorrhagic enteritis in a single baboon (Papio cynocephalus) (Boncyk et al., 1972), and was isolated from 11 baboons with chronic diarrhea (Tribe et al., 1979). The organisms have not been reported isolated from other species of apes, including orangutans (Pongopygmeus). In all species susceptible to Campylobacter spp. infection, the major route of transmission is fecal-oral. Fowl are important reservoirs of the organisms, and domestic poultry are known to play a significant role in human infections (Harris et al., 1986; Pazzaglia et al., 1993; Glass et al., 1983). Although consumption of undercooked poultry and foods contaminated in the market are important sources of human infection (Blaser et al., 1983), keeping fowl inside the home has also been shown to be a risk factor for Carnpylobacter-associateddiarrhea (Pazzaglia et al., 1993), suggesting transmission from fowl can occur indirectly through environmental exposure. Except for zoonotic outbreaks involving humans, transmission of campylobacters from animal-to-animal has not been documented, but probably occurs frequently. Through an international cooperative agreement, 10 orangutans were recently returned to Indonesia from Taiwan where they had been retained by private owners in their residences, under a variety of living conditions (Campbell, 1991). The 6 male and 4 female apes (aged 18 months to 5 years) were quarantined in a dedicated primate facility adjacent to the Ragunan Zoo in South Jakarta until their relocation to the Tanjung Puting National Park in Kalimantan. Shortly after their arrival in Jakarta, the animals began experiencing recurrent bouts of diarrhea. After unsuccessful treatment by primate facility personnel, the U.S. Naval Medical Research Unit No. 2 (NAMRU-2) was asked to assist in the diagnosis and treatment of the sick animals. This article reports the findings related to the etiology, clinical manifestations, probable sources of infection, and treatment of these 10 primates.

Materials and methods

Outbreak investigation and specimens General The 10 orangutans arrived in Jakarta in November, 1990, and were immediately placed in the primate facility adjacent to the Ragunan Zoo. The animals remained housed in this facility during all aspects of this study. Rectal swabs or fresh stool were acquired from each of the orangutans on 4 occasions, at approximately 3 week intervals, and tested for common bacterial and parasitic enteropathogens. Initial specimens Initial rectal swab fecal samples, which had been preserved in Cary-Blair medium (Cary and Blair, 1964), were delivered by primate facility personnel to NAMRU-2 for laboratory testing (February 7). No clinical information for individual animals was provided with this initial group of specimens. All but one of the animals reportedly had suffered recurrent bouts of diarrhea since their arrival, and the majority of the animals (number not specified) were diarrheic at the time when specimens were obtained. Primate facility personnel were notified of the laboratory results (3 animals Campylobacter-positive) and provided a recommendation for a 7-day course of oral erythromycin (Morton et al., 1981; Tribe et al., 1979; McNulty, 1987). February 26 site visit Three weeks after our first contact with facility personnel, NAMRU-2 was again contacted and informed that the animals continued to experience recurrent episodes of diarrhea. Because the recommended erythromycin 'had not been available' (for reasons unclear to the authors), primate facility personnel had treated the animals with triple sulfa (250 mg t.i.d, per os for 3 days; 34 68 mg/kg/day), then metronidazole (125 mg/ml elixir; 5 ml b.i.d, per os for 2 days; 59-114 mg/kg/day). The last treatment had been given 10 days before, and the handlers reported there had been no significant improvement following either treatment. A field team visited the facility and a second group of fecal specimens were obtained from all 10 orangutans. Although detailed physical examinations were not performed, 6 orangutans were observed to be diarrheic at the time of the visit; 3 others had a recent history (less than 48 h) of diarrhea. A team of 4 handlers were quartered in the same facility, providing 24 h care to the orangutans; when interviewed, none recalled having had recent diarrhea, fever, or other illness. A survey was made of the orangutan facility; husbandry practices and clinical histories were determined by interviewing the animal caretakers. The orangutans were housed in individual cages in a partially enclosed rectangular tile-floored area approximately 10 × 10 m, which was open to a driveway and yard area on one side. Cages were large, stainless steel primate cages of the commercial variety, and were thoroughly cleaned once per week with disinfectant and abundant water. The cage area floor was scrubbed and disinfected once per day; feces deposited on the floor were cleaned up almost immediately with a squeegee and disinfectant. Sterile gauze pads, moistened with sterile phosphate buffered saline (PBS) were used to swab floor, cage, and food preparation surfaces, and preserved for bacteriological testing. Animals were allowed out of their cages during the day while under the supervision

of the caretakers. During these periods, animals were free to exercise and play in the immediate vicinity of the cages, sometimes venturing just outside the cage area, onto the driveway or yard. In the evenings, the animals did not necessarily return to the same cages they had previously occupied. Only the 2 senior handlers prepared the animals' food and water. The animals' drinking water was the same commercially bottled water (sealed 20 1. plastic containers) commonly used for local human consumption. Three 50 ml samples of the animals' drinking water were obtained for bacteriological testing. Fresh food was purchased daily, and an abundant supply was on hand at the time of the visit; fruits and vegetables were cleaned and soaked in a bleach solution (estimated to be about 10% household bleach) and kept in a rodent-free storage area in the house until feeding time. The orangutans had no direct contact with other animals. However, the neighbors' chickens, which were kept in the yard area adjacent to the facility, were observed to sometimes wander into the cage area. Numerous fresh chicken droppings were seen on the driveway, just outside the cage area. One of these droppings was obtained for bacteriological testing. Treatment with oral erythromycin was again recommended, along with minor modifications of husbandry practices. The most significant of these were: Do not allow the orangutans outside the cage area. Surface disinfect cages each time the animals are released, so that returning animals always enter freshly disinfected cages. Disinfect the entire cage area floor twice per day, immediately before the mid-day and evening meals. Establish a physical barrier to keep chickens out of the compound, especially the cage area.

March 15 site visit Two weeks later, we were notified by primate facility personnel that the recommended changes in care and management had been instituted at the orangutan facility, and the health of the animals had temporarily improved. But because the animals' condition had initially improved, the handlers had decided to discontinue treatment, and the course of erythromycin had not been completed by any of the primates. Now, several of the animals were once again sick with diarrhea. This prompted a second visit to the facility by the field team, and collection of either fresh feces or rectal swab samples from each of the 10 orangutans. Two of the animals were found to have watery diarrhea (Imelda, Made); another animal had recently had a 3-day episode (Otong). Thirteen houseflies (Musca domestica) were obtained from the caging area and pooled in sterile PBS for bacteriological examination. During this second visit, facility personnel were provided with adequate erythromycin oral suspension for a full course of treatment for all animals (40 mg/ml; Kalbe Farma Kalthrocin; 7 days); dosage was equivalent to that recommended for children (30-50 mg/kg/day). To ensure the full course was administered, treatment was monitored. Followup-site visit Three weeks later, a followup visit was made to determine progress. All recommended procedures were still in place. All animals had completed at least 7 days of erythromycin treatment and all appeared healthy. The handlers indicated that the

diarrhea had ceased within 2 days after beginning antibiotic therapy, with no recurrences. Rectal swabs were obtained from all 10 orangutans and cultured for Campylobacter spp.

Laboratory All specimens were processed within 2 h of collection. Rectal swabs were preserved in Cary-Blair medium for transport; fresh fecal samples were collected in sterile containers; environmental samples were placed in sterile containers, suspended in sterile PBS, or placed in Cary-Blair medium, depending on the type of specimen. Fecal specimens were tested for common bacterial enteropathogens using conventional bacteriological methods, and for intestinal parasites by microscopic examination after filtration, centrifugation, and ether extraction (Balows et al., 1991). For bacteriological testing, direct inoculations of fecal and environmental specimens were made onto standard commercial enteric media (Difco Laboratories, Detroit, MI), including Hektoen agar, thiosulfate-citrate-bile-sucrose agar (TCBS), MacConkey agar (MAC), salmonella-shigella agar (SS) and Skirrow's medium (SKM) (Skirrow, 1977). Following direct inoculations, swabs were placed in alkaline peptone water (pH 8.6) (APW) and gram negative broth (GNB) enrichments. Primary cultures (except on SKM) and enrichments were incubated aerobically overnight at 36 ± 1°C. Cultures on SKM were incubated for 48 h in a microaerophilic environment at 42 _+1°C for isolation of Campylobacter spp. After overnight incubation, APW enrichments were subcultured onto TCBS and MAC, GNB enrichments were subcultured onto MAC and SS, then subcultures were incubated overnight at 36 _+1°C. Suspect colonies were identified using conventional biochemical and serological methods (Balows et al., 1991), augmented with the API 20E identification system (Analytab, Plainview, NY) when indicated. Species identification of Campylobacter spp. isolates was by growth at 42°C, catalase production, hippurate hydrolysis, and resistance to cephalothin (30 ug) (Cornick and Gorbach, 1988). Surface swabs were processed by twirling in sterile PBS, then inoculating the bacterial suspension onto direct and enrichment cultures as described above. The houseflies, obtained from the cage area and placed in PBS, were homogenized, then cultured for Campylobacter spp. in the same manner as fecal specimens. The 3 potable water samples were tested for common bacterial enteropathogens (but not Campylobacter spp.) by filtering each of the samples through a 0.45 u sterile filter, then placing the filter onto blood agar medium. Campylobacter spp. isolates were preserved in 20% glycerol-trypticase soy broth (-85°C), then later recovered and tested for their susceptibility to 12 antibiotics by the disk diffusion method (Balows et al., 1991). To confirm results, hippurate hydrolysis and antibiotic susceptibility tests were repeated for all strains. Results

Bacteriology Initial specimens Three of the 10 orangutan fecal cultures yield C. coli. No other bacterial or parasitic enteropathogens were detected.

First on-site visit Laboratory findings for the second group of specimens indicated that the animals had continued to be infected with Campylobacter spp.; 10 of 10 fecal specimens were culture-positive. However, only 4 of the 10 isolates were C. coli, and the remaining 6 were C. jejuni. Shigella sonnei was isolated from one of the animals (Lucy), who was the only orangutan with no recent history of diarrhea. No pathogenic enteroparasites were detected. C. coli was isolated from chicken feces obtained from the driveway just outside the cage area, and C. jejuni was detected in an orangutan fecal specimen retrieved from the floor (host uncertain). Surface swab specimens were negative for Campylobacter spp. and other bacterial enteropathogens; the 3 potable water samples were also negative, but were not cultured for Campylobacter spp.

Second on-site visit C. jejuni was isolated from rectal swabs from 6 of the l0 orangutans, and from the pool of 13 houseflies obtained from the perimeter of the cage area.

Followup visit All of the 10 fecal specimens tested were negative for bacterial and parasitic enteropathogens.

Antibiotic susceptibilities All of the 22 Campylobacter spp. isolates were resistant to cephalothin, and susceptible to chloramphenicol, furandantin, gentamicin, colistin, streptomycin and erythromycin. Based on species-specific susceptibility results for antibiotics which showed variable inhibitory effects on different Campylobacter strains (ampicillin, tetracycline, trimethoprim-sulfamethoxazole, triple sulfa, kanomycin, and neomycin), C. coli isolates were categorized into 4 groups (A-D), and C. jejuni isolates into 4 groups (E-H) (Table 1). Significantly, all isolates from the third set of specimens (March 15) were C. jejuni, and had the same antibiotic susceptibility profile (H). This particular profile had not been previously detected from animal or environmental specimens.

Clinical Only one orangutan (Lucy) did not develop diarrhea while housed at the orangutan facility, even though C. jejuni and 5;. sonnei were isolated from this animal's feces. The most seriously ill animal observed during the site visits (Charlie) appeared mildly dehydrated, somewhat lethargic, and could be classed as having a moderately-severe secretory-like diarrhea, with an increased frequency of watery, mucoid stools having greater than usual volume. Other animals were mild-to-moderately diarrheic and had no noticable signs of dehydration. Stools varied from loose to liquid, usually with mucus. None of the animals had bloody stools. Stool frequencies were increased, but usually of normal volume. The duration of individual episodes ranged from 2 days, to over 2 weeks (Romeo).

7 TABLE 1 Disk diffusion antibiotic susceptibilities of Campylobacter spp. isolates from fecal and environmental specimens obtained from a colony of orangutans (Pongo pygmaeus) Source of isolatea

Host status

Species

Date of Antibiotic susceptibilities isolate Campylobacter spp. isolatesa'b Amp Tet Tmp-smx Sss Kan Neo Profile

PP/F (Made) PP/F (Imelda) PP/F (Manis) PP/F (Grence) PP/F (Dede) PP/E (Made) PP/F (Otong) Chicken feces PP/E (floor) PP/F (Manis) PP/F (Budha) PP/F (Charlie) PP/F (Lucy) PP/F (Imelda) PP/F (Romeo) Houseflies PP/F (Otong) PP/F (Imelda) PP/F (Lucy) PP/F (Manis) PP/F (Charlie) PP/F (Romeo)

n.r. a n.r. n.r. Recent diarrhea Diarrheic Diarrheic Recent diarrhea Environment Environment Recent diarrhea Diarrheic Diarrheic Healthy Diarrheic Diarrheic Environment Recent diarrhea Diarrheic Healthy Healthy Healthy Healthy

C. coli C coli 62 coli C. coli C. cob C. coli C. coli C coli 62 jejuni 62 jejuni 62 jejuni C jejuni C. jejuni C. jejuni C jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni

2 07 2-07 2-07 2-26 2-26 2 26 2-26 2-26 2-26 2 26 2 26 2-26 2-26 2 26 2 26 3-15 3 15 3-15 3-15 3 15 3-15 3-15

R R R R R R R R S S S S S S S R R R R R R R

R R R R S S S S S S S S S S S S S S S S S S

S S R S R R R R S R R R R R R R R R R R R R

R S R R R R R R S S S S S R R R R R R R R R

S R R S S S S S S S S S S S S S S S S S S S

S R R S S S S S S S S S S S S S S S S S S S

(a) (b) (c) (a) (d) (d) (d) (d) (e) (f) (f) (f) (f) (g) (g) (h) (h) (b) (h) (h) (h) (h)

Strains having the same antibiotic susceptibility profile (last column) yield equivalent results in all individual tests. aAmp, ampicillin; Tmp-smx, trimethoprim-sulfa-methoxazole; Tet, tetracycline; Kan, kanamycin; Sss, triple-sulfa; Neo, neomycin; PP, Pongo pygmaeus; F, fecal specimen; n.r., not recorded; R, resistant; S, susceptible. bAll strains were resistant to cephalothin and susceptible to chloramphenicol, furadantin, gentamicin, colistin, streptomycin, and erythromycin.

Discussion T h e c u r r e n t o u t b r e a k o f d i a r r h e a a m o n g the c o l o n y o f 10 o r a n g u t a n s was c h a r a c t e r i z e d by p e r s i s t e n t a n d r e c u r r i n g e p i s o d e s o f w a t e r y , m u c o i d d i a r r h e a , w i t h o u t evidence of dysentery. The outbreak was typical of a closed epidemic of infectious d i a r r h e a in w h i c h c o n t i n u o u s t r a n s m i s s i o n occurs, e i t h e r f r o m a c o n s t a n t o r i n t e r m i t t e n t e n v i r o n m e n t a l source, o r f r o m o r g a n i s m s b e i n g t r a n s f e r r e d f r o m h o s t to h o s t via a f e c a l - o r a l r o u t e , p r o v i d i n g c o n t i n u o u s cycles o f i n f e c t i o n - r e c o v e r y - r e i n f e c t i o n . T h e m o s t p r o b a b l e c a u s e o f the illnesses was m u l t i p l e i n f e c t i o n s b y C a m p y l o b a c t e r spp., w h i c h w e r e i s o l a t e d f r o m all o f t h e a n i m a l s . S. sonnei w a s i s o l a t e d f r o m o n e o f t h e p r i m a t e s , b u t the i s o l a t i o n w a s f r o m feces o f a c o n s i s t e n t l y h e a l t h y o r a n g u t a n , m a k i n g it u n l i k e l y t h a t this a g e n t c o u l d h a v e b e e n r e s p o n s i b l e f o r t h e c o n c o m i t a n t disease o c c u r r i n g in t h e o t h e r a n i m a l s . R e s u l t s f r o m e x a m i n a t i o n s o f s t o o l s f o r diarrheagenic parasites were negative.

The isolation of 8 distinct strains of Campylobacter spp. (as characterized by antibiotic susceptibility profiles) from this closed population, suggested new infections were being introduced while other Campylobacter strains were causing active disease. Although the occurrence of orangutan-to-orangutan transmission was almost certain, it is doubtful that all 8 of the strains were introduced to the colony simultaneously and then continued to circulate among the infected animals. This was apparent from the major antibiotic susceptibility shifts that occurred between each visit to the colony. Drinking water and food appeared to be unlikely sources of infection. During the first site visit, vegetables were observed being scrubbed, then soaked, in a household bleach solution. The bottled water was of the same brand which has been tested on numerous occasions in our laboratory for coliforms and other bacterial contaminants; in all instances, the water samples have been within limits for human consumption. Infection transmitted from the animal handlers seemed unlikely because interviews revealed no recent history of illness among any of them. In subsequent conversations during later visits to the facility, the continued healthy state of the handlers was apparent and uninterrupted. When asked, the handlers were unwilling to provide stool specimens or rectal swabs, and so none were obtained for culture. The most likely source of continuous infection by multiple strains of campylobacters was fecal contamination from the poultry entering the orangutan housing area. Although only one chicken fecal sample was tested, it was noteworthy that the C. coli isolate recovered had the same antibiotic profile as strains detected in feces from 3 of 4 culture-positive orangutans who had current or recent illness. Unfortunately, no additional chicken feces were tested because the association was not realized until after the outbreak, when isolates were further characterized in regard to antibiotic susceptibilities. Our suggestions had included the installation of a wire barricade to keep the fowl away from the cage area, but the expense of installing such a fence had been prohibitive. Instead, an individual was hired to prevent the birds from coming into the area by waving them away when they got too close. The handlers assured us that this person had been effective in keeping the chickens away from the orangutans. Of interest was that for a short time after the chickens were prevented from entering the cage area, the animals reportedly recovered, and then were again stricken with diarrhea. During this new wave of infections, all 7 C. jejuni isolates obtained (March 15) were of the same antibiotic profile (H). This was in contrast to the first samples obtained (February 7), when the 3 C. coli isolates had different profiles (A, B, and C), and the second group of samples (February 26), in which the 12 isolates fell into 5 profile groups (A, D, E, F, and G). This provided circumstantial evidence that a single triple-sulfa-resistant strain of C. jejuni ( H ) may have been introduced after or during the partial course of treatment with triple-sulfa and metronidazole. It was also the same strain isolated from houseflies. Because direct contact with chickens (and their feces) by the orangutans had been effectively eliminated during this period, a possible alternative source of new infection was from the houseflies, which had unhindered access to chicken feces, the animals, and their food. However, there is also the possibility that one or more of the animals were colonized with this strain earlier, and the strain was not detected until it became the

predominant strain (through antibiotic selective pressure) by the time of the third visit. That this strain of C. jejuni was then transmitted to other primates (via fecaloral) and flies (via direct contact with feces), could not be excluded. Although no blood could be obtained for immunological studies, the recurrent episodes of disease, probably caused by different strains of C. coli and/or C. jejuni in an single host, suggest that cross-strain protective immunity may not be elicited in an individual orangutan from a single infection. This was evidenced by Made, who was infected with 2 different strains of C. coli, and Manis, Charlie, Lucy, Imelda, and Romeo, who were individually colonized with different strains of C. jejuni, at different points in time. Made and Imelda were both diarrheic at the time when the second strain was detected. Instead, it seems that back-to-back episodes of CampyIobacter-associated diarrhea can occur, so long as the infecting strains are [antigenically] distinct. This appears to be similar to what occurs in h u m a n populations, where protective immunity may be serotype-specific, and cross-strain protection is only conferred on the human host after multiple exposures over extended periods of time (Blaser et al., 1985, 1983, 1986; Taylor et al., 1988). We have described an outbreak of persistent, recurring diarrhea among orangutans, most likely caused by multiple transmissions of different C. coli and C. jejuni strains. Infection probably occurred through several mechanisms, including direct host-to-host fecal-oral transmission, consumption of foods or water directly contaminated with chicken feces, and vectored transmission, in which c o m m o n houseflies may have provided a contamination link between chicken feces and consumables. Antibiotic susceptibility profiles of Campylobacter spp. isolates offered evidence that numerous different Campylobacter strains were circulating in a relatively small vicinity, and that a single C. coli or C. jejuni infection did not confer protective immunity against exposure to heterologous strains, even within the same species.

Acknowledgements Thanks and appreciation are extended to the Orangutan Foundation and the Indonesian government for their continued efforts in helping to ensure these beautiful and docile animals continue their quiet existence in the rain forests of Indonesia. Thanks are also due to D. Temple for critical review of the manuscript and useful editorial comments. This work was supported by the U.S. Naval Medical Research and Development Command, Bethesda, MD, USA, work unit number 627871/6.2/275/75.

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