Infectious Agent and Immune Response Characteristics of Chronic Enterocolitis in Captive Rhesus Macaques

INFECTION AND IMMUNITY, July 2003, p. 4079–4086 Vol. 71, No. 70019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.7.4079–4086.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Infectious Agent and Immune Response Characteristics of ChronicEnterocolitis in Captive Rhesus Macaques

Karol Sestak,1* Christopher K. Merritt,1 Juan Borda,1 Elizabeth Saylor,1 Shelle R. Schwamberger,1Frank Cogswell,1 Elizabeth S. Didier,1 Peter J. Didier,1 Gail Plauche,1 Rudolf P. Bohm,1

Pyone P. Aye,1 Pavel Alexa,2 Richard L. Ward,3 and Andrew A. Lackner1

National Primate Research Center, Covington, Louisiana1; Department of Bacteriology, Veterinary Research Institute,Brno, Czech Republic2; and Division of Infectious Diseases, Cincinnati Children’s Hospital, Cincinnati, Ohio3

Received 25 November 2002/Returned for modification 26 February 2003/Accepted 26 March 2003

Chronic enterocolitis is the leading cause of morbidity in colonies of captive rhesus macaques (Macacamulatta). This study’s aim was to identify the common enteric pathogens frequently associated with chronicenterocolitis in normal, immunocompetent rhesus monkeys and to elucidate the influence of this clinicalsyndrome on the host immune system. We analyzed the fecal specimens from 100 rhesus macaques with orwithout clinical symptoms of chronic diarrhea. Retrospective analysis revealed an increased incidence ofCampylobacter spp. (Campylobacter coli and Campylobacter jejuni), Shigella flexneri, Yersinia enterocolitica, ade-novirus, and Strongyloides fulleborni in samples collected from animals with chronic diarrhea (P < 0.05). Thepresence of additional enteric pathogens, such as Escherichia coli, carrying the eaeA intimin or Stx2c Shigatoxin virulence genes, Balantidium coli, Giardia lamblia, Enterocytozoon bieneusi, and Trichuris trichiura wasfound in all animals regardless of whether diarrhea was present. In addition, the upregulation of interleu-kin-1� (IL-1�), IL-3, and tumor necrosis factor alpha cytokine genes, accompanied by an increased presenceof activated (CD4� CD69�) T lymphocytes was found in gut-associated lymphoid tissues collected fromanimals with chronic enterocolitis and diarrhea in comparison with clinically healthy controls (P < 0.05).These data indicate that chronic enterocolitis and diarrhea are associated, in part, with a variety of entericpathogens and highlight the importance of defining the microbiological status of nonhuman primates used forinfectious disease studies. The data also suggest that chronic colitis in rhesus macaques may have potential asa model of inflammatory bowel disease in humans.

A high incidence of chronic enterocolitis associated with thepresence of opportunistic and/or obligate pathogens known tocause disease of the gastrointestinal tract has been recordedfrom colonies with captive rhesus monkeys (13, 25, 28). Incolonies of nonhuman primates, recurring diarrhea is the lead-ing cause of animal morbidity requiring veterinary care (13,25). The success of the rhesus macaque model of AIDS and theidentification of the intestinal immune system as a primarytarget of human immunodeficiency virus and simian immuno-deficiency virus (SIV) has resulted in increased demands formicrobiologically and genetically defined animals. The needfor pathogen defined rhesus monkeys has reached critical pro-portions (9, 10, 27). At present, most rhesus monkey specific-pathogen-free colonies are defined by negative testing for fourviral agents (SIV, type D simian retrovirus, simian T lympho-tropic virus type 1, and simian herpes B virus). However, AIDSresearch focusing on opportunistic infections, novel vaccinestrategies, immune modulating pharmaceutical agents andgene therapy approaches may require that rhesus monkeys befree of additional viruses, protozoa, and bacteria. Developingstrategies to prevent or treat enteric infections and elucidatetheir role, or lack thereof, in AIDS enteropathy and diseaseprogression is one objective of research at the Tulane NationalPrimate Research Center (TNPRC) (32, 33). Thus, the aim of

the present study was to determine the incidence of infectiousmicroorganisms commonly associated with symptoms ofchronic enterocolitis in captive rhesus macaques, thereby im-proving the research value of this animal model. In addition,we evaluated the level of general immune response indicatorsin regard to systemic and mucosal lymphoid tissues from theseanimals by measuring the expression of CD69 and CD45RA byCD4� and CD8� T lymphocytes and RNA expression of 23cytokine genes. We hypothesized that chronic inflammation ofthe colon in rhesus monkeys might not only clinically but alsoimmunologically resemble the inflammatory bowel disease(IBD) of a human patient.

MATERIALS AND METHODS

Rhesus macaques: categories and fecal sample collection. One hundred rhesusmacaques (Macaca mulatta) of either sex were examined as they were admittedto veterinary clinic at TNPRC in between January and June 2002. The ages ofstudied animals ranged from 6.5 months to 24.5 years. All animals originatedfrom the TNPRC and were housed in biosecurity level 2 (BL2) in accordancewith standards of the Guide for the Care and Use of Laboratory Animals and theAssociation for Assessment and Accreditation of Laboratory Animal Care. Onlysimian retrovirus (SIV, simian retrovirus, and simian T lymphotropic virus)- andtuberculosis-negative animals were used. Investigators adhered to the Guide forthe Care and Use of Laboratory Animals prepared by the National ResearchCouncil. Fifty animals had no clinical symptoms of diarrhea (surgery-relatedadmissions), and the other fifty animals showed symptoms of chronic diarrhea atthe time of sample collection (Table 1). Chronic diarrhea was defined by themanifestation of multiple outbreaks of clinical diarrhea over a period of at least1 month. In general, every animal with or without a history of diarrhea that waspresented to the veterinary clinic at TNPRC was clinically monitored on a dailybasis. To obtain stools, cage pans were cleaned in the morning and stools were

* Corresponding author. Mailing address: Division of Microbiologyand Immunology, Tulane National Primate Research Center, 18703Three Rivers Rd., Covington, LA 70433. Phone: (985) 871-6409. Fax:(985) 871-6248. E-mail: [email protected].

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collected in the afternoon. Collected samples were made into 50% suspensionswith phosphate-buffered saline (PBS; pH 7.4) and divided into aliquots for virus,bacterium, and parasite screening. Samples to detect bacteria and parasites wereprocessed on the day of sample collection. Samples for viral analysis were storedfrozen at �80°C until tested.

Histopathological evaluation and tissue sample collection. Tissues from sixrhesus macaques were collected. Three animals with clinical symptoms of chronicdiarrhea (J079, CJ41, and CJ51) and three age-matched control animals withoutclinical symptoms of diarrhea (T080, DE22, and V176) were randomly selected.All six animals received a complete necropsy and histopathologic examination.Samples of systemic tissues (spleen), gut-associated lymphoid tissues (mesentericlymph nodes), and the gastrointestinal tract had been collected immediately aftereuthanasia with an intravenous overdose of pentobarbital. The small and largeintestines were removed and separated into the duodenum; jejunum; ileocecaljunction; proximal, middle, and distal colon; and rectum. Tissues were fixed in10% neutral buffered formalin, routinely processed for histopathologic exami-nation, sectioned at 6 �m, and stained with hematoxylin and eosin (H&E). Inaddition, the spleens and mesenteric lymph nodes were used for (i) isolation ofmononuclear cells and flow cytometry as previously described (31, 38) and (ii)frozen at �80°C for later extraction of total RNA.

Detection of gastrointestinal pathogens. The presence of a variety of viral,bacterial, and parasitic agents known to infect nonhuman primates was exam-ined.

Viral agents. A commercially available immunoassay (Dako, Cambridgeshire,United Kingdom) that is capable of detecting the presence of any of the 49known adenovirus serotypes (product code K6021) was used according to themanufacturer’s instructions to examine the presence of adenoviruses in 100 ofthe fecal specimens. In addition, all samples were tested by enzyme-linkedimmunosorbent assay for the presence of rotavirus antigens at the CincinnatiChildren’s Hospital as described previously (23).

Bacterial agents. In order to detect the presence of common bacterial patho-gens, the following method was used. Stool samples were plated on (i) xylose-lysine-deoxycholate agar (BBL, Cockeysville, Md.), (ii) Hektoen enteric agar(BBL), (iii) eosin methylene blue agar (BBL), (iv) Campylobacter selective agar(BBL), (v) Yersinia selective agar (Oxoid, Ltd., Basingstoke, England), and (vi)Yersinia enrichment broth (Oxoid). Plates 1, 2, and 3 were examined after 18 to24 h of incubation at 37°C. Colonies indicative of Salmonella sp. or Shigella sp.were subcultured to triple sugar iron agar (BBL), incubated for 18 to 24 h at37°C, and typed with the appropriate antisera. Colonies suggestive of Escherichiacoli were identified and tested for the presence of enteropathogenic Escherichiacoli (EPEC) and enterotoxigenic Escherichia coli strains at the Veterinary Re-search Institute, Brno, Czech Republic, and Tulane University Health SciencesCenter, respectively, as described previously (6, 7). Multiplex PCR was used totest for the presence of genes responsible for eaeA intimin and Stx1, Stx2, andStx2c Shiga toxin as described previously (24). Positive strains were tested for theproduction of Vero toxins (6). Plate 4 was incubated for 48 h at 42°C in anatmosphere of 10% carbon dioxide and 5% oxygen, with a balance of nitrogen.Confirmation of Campylobacter identification was done by Gram stain and de-termining susceptibility to nalidixic acid and cephalothin. The hippurate test wasused to differentiate Campylobacter coli and Campylobacter jejuni (14). Plate 5was incubated at room temperature for 48 h. Colonies indicative of Yersinia weresubcultured to TSIA slants, urea broth, phenylalanine slants, and analyticalprofile index (bioMerieux, Marcy l’Etoile, France) identification panels. TheYersinia cold broth (plate 6) remained in the refrigerator (4°C) for 5 to 7 days.Subsequently, it was subcultured onto a Yersinia selective agar (Oxoid) plate, andthe procedure for plate 5 was repeated.

Parasitic agents. For the detection of parasitic agents, a direct fecal smear,followed by a concentration technique, was used (1). Briefly, by the use of an

applicator stick, 2 mg of stool was added to a drop of PBS (pH 7.4) on a slide.After thorough mixing, a 22-by-22-mm coverslip was added. The motility of theorganisms was observed under a light microscope (magnification, �40 to �100).In addition to the direct smear, a flotation method was performed as describedpreviously (1), and the preparations were examined by light microscopy. Fordetection and characterization of microsporidia (Enterocytozoon bieneusi), cal-cofluor staining to screen the samples and nested PCR to confirm the results withEnterocytozoon bieneusi primers were used as previously described (16, 33).

Cytokine gene expression. Total RNA was isolated from 3 g of mesentericlymph node or spleen tissues and tested for the expression of 23 commoncytokine genes (interleukin-1� [IL-1�], IL-1�, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12�, IL-12�, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,gamma interferon [IFN-�], tumor necrosis factor alpha [TNF-�], and TNF-�)and internal standard control genes (�-actin, GAPDH [glyceraldehyde-3-phos-phate dehydrogenase], and pUC18) by using a Nonrad-GEArray kit (SuperAr-ray, Inc., Bethesda, Md.). Briefly, biotinylated cytokine DNA probes were con-structed from the total RNA templates by reverse transcription of mRNA byusing a cytokine-specific primer mix supplied by the manufacturer, biotin-16-dUTP (Promega, Madison, Wis.) and Moloney murine leukemia virus reversetranscriptase (Promega). The labeling reaction was stopped after 4 h, and theprobes were denatured and added to a hybridization solution. During the label-ing reaction, a gene expression array cytokine membrane (GEArray membrane)was treated by prehybridization with salmon sperm DNA (Invitrogen, Inc., Carls-bad, Calif.) to prevent nonspecific binding. The GEArray membrane was thenhybridized overnight with the probe solution in a sealed plastic bag. Afterhybridization, the probe solution was washed off and the GEArray membranewas incubated with a 1:4,000 alkaline phosphatase-streptavidin solution (sup-plied with GEArray) and subsequently with CDP-Star color substrate solutionfor the visualization of the hybridization (spot) reaction. A sheet of X-ray film(Kodak, Rochester, N.Y.) was used to develop an image of the membrane.Relative mRNA expression was calculated from the films via local backgroundcorrected spot densiometry measurements with ScanAlyze v2.44 software (Stan-ford University, Stanford, Calif.) and comparison with the �-actin (positive) andpUC18 (negative) controls built into the array.

Flow cytometry. Spleen and mesenteric lymph nodes were used for isolation ofmononuclear cells as previously described (31). Gating was set based on theCD3� “bright” T-lymphocyte population as determined by single-color staining.Proportions of CD69� (activated T lymphocytes) and CD45RA (naive T lym-phocytes) in CD4� and CD8� (T helper and cytotoxic/suppressor lymphocytes)populations were assessed by four-color flow cytometry. The combination ofCD69, CD45RA, CD4, and CD8 was used for the mononuclear cells isolatedfrom mesenteric lymph nodes and spleens as described previously (31, 33).Briefly, the extracted, purified, and stained mononuclear cells were fixed in PBS(pH 7.4) containing 1% paraformaldehyde (Sigma, St. Louis, Mo.). A FACS-Calibur flow cytometer with CellQuest software (Becton-Dickinson, FranklinLakes, N.J.) was used to perform the quantitation of naive and activated CD4�-and CD8�-T-lymphocyte populations.

Statistical analysis. The prevalence of each infectious agent in the chronicdiarrhea group was compared to its prevalence in the clinically healthy group byusing the chi-square test. The effect of multiple bacterial, viral, and parasiteinfections, specifically adenovirus, Campylobacter spp., Shigella flexneri, Yersiniaenterocolitica, and Strongyloides fulleborni, on the health status (clinically healthyversus chronic diarrhea) was statistically analyzed by using the chi-square distri-bution test. The cytokine gene expression and T-lymphocyte populations werecompared between the two groups described above by a paired-sample Studentt test with two-tailed distribution.

RESULTS

Histopathology findings. A range of histopathologicalchanges were observed in rhesus macaques with clinical symp-toms of chronic diarrhea that were euthanized (Fig. 1). Themost typical and prominent lesions included the infiltration ofcolon lamina propria with inflammatory cells, such as lympho-cytes, neutrophils, and plasma cells; the loss of goblet cells;crypt dilation; amyloidosis; and the accumulation of necroticdebris in the lumen of the crypts. The colon and terminal ileumwere the most affected parts of chronically inflamed intestines.Other parts of the intestinal tract, such as the duodenum,

TABLE 1. Clinical summary of 100 rhesus macaques from whichsamples were obtained

Group (n) Mean age(yr) � SD

Male/femaleratio

Clinicaldiarrhea

(%)

Mean stoolconsistencya

� SD

History ofdiarrhea

(%)

Clinicallyhealthy (50)

6.2 � 6.2 0.32 0 1 � 0 32

Chronicdiarrhea (50)

6.0 � 5.8 0.42 100 3.8 � 0.4 100

a Stool consistency was assigned based on a scale from 1 to 5: 1, normal; 2,pasty; 3, semiliquid; 4, watery; 5, watery with the presence of blood.

4080 SESTAK ET AL. INFECT. IMMUN.

FIG. 1. H&E-stained tissue sections of small and large intestine tissues from selected animals with symptoms of chronic diarrhea (J079, CJ41, andCJ51) and a clinically normal control (T080). (A) Normal colon (T080) with straight crypts, the presence of goblet cells, and lamina propria with a fewmononuclear cells. (B) Chronic colitis (J079). The lamina propria is densely infiltrated by mononuclear cells. (C) Chronic colitis (CJ51). The loss of gobletcells from mucosal epithelium and the presence of Balantidium coli (BC) is shown. (D) Active chronic colitis (CJ51). Moderately dense infiltration oflamina propria by mononuclear cells, crypt dilatation, and crypt abscess with accumulation of granulocytes and necrotic debris in the lumen of the glandcan be seen. (E) Duodenum, chronic enteritis (CJ51). Inflammatory cells are present in the lamina propria with inflammation confined to the mucosa.(F) Ileocecal junction, severe chronic ulcerative ileocolitis (CJ41). Both acute and chronic inflammatory cells are present. Numerous neutrophils arepresent in the ulcer. Lymphocytes, neutrophils, plasma cells and fibroblasts are present in the lamina propria.

4081

ileum, and cecum, were also affected with the accumulation ofinflammatory cells, mostly in the lamina propria (Fig. 1).

Gastrointestinal agents identified. A variety of infectiousagents were identified in both clinically healthy animals andanimals with chronic diarrhea. Microorganisms and/or entericpathogens that were detected more frequently (P 0.05) inanimals with chronic diarrhea than in clinically healthy animalsincluded adenovirus; enteric bacteria including Campylobacterspp. (Campylobacter coli and Campylobacter jejuni species),Shigella flexneri, and Yersinia enterocolitica; and the parasiticnematode Strongyloides fulleborni (Fig. 2). In contrast, entericpathogens detected in all animals without being significantlymore prevalent in macaques with chronic diarrhea includedEPEC carrying the eaeA intimin or Stx2c Shiga toxin virulencegenes; the protozoan parasites Balantidium coli and Giardialamblia; the helminth Trichuris trichiura; and the microspo-ridium Enterocytozoon bieneusi (Fig. 2). The effect of multiplebacterial, viral, and parasite infections, specifically adenovirus,Campylobacter spp., Shigella flexneri, Yersinia enterocolitica, andStrongyloides fulleborni, on the health status (i.e., clinicallyhealthy animals versus animals with chronic diarrhea) was sta-tistically examined. The cumulative effect of these infectiousagents was found to be significant at a P value of 0.002 (df 4). The production of verotoxin was detected by cytotoxicityassay in two of seven of Stx2c Escherichia coli strains. Norotavirus was detected in any of the animals tested. In additionto the enteric pathogens described above, we identified up to a90% incidence of ameboid protozoa (Entamoeba coli and Io-doamoeba spp.) and a 26% incidence of protozoan flagellates(Trichomonas and and Chilomastix spp.) regardless of whetherthe animals did or did not have chronic diarrhea.

T-lymphocyte populations. Four-color flow cytometry wasused to determine the distribution of activated and/or naiveCD4� and CD8� T lymphocytes in gut-associated lymphoid

tissues (mesenteric lymph nodes) and spleens. In clinicallyhealthy animals both gut-associated lymphoid tissues andspleen CD4� and CD8� T lymphocyte populations containeda higher proportion of naive (CD45RA�) cells (48.5% � 8.2%and 77.7% � 8.9%, respectively) and a lower proportion ofactivated (CD69�) cells (12.3% � 4.0% and 4.7% � 2.6%)compared to animals that exhibited clinical symptoms ofchronic diarrhea (Table 2 and Fig. 3). Statistically significantelevations (P 0.05), however, were only found in CD4�

CD69� T lymphocytes in the mesenteric lymph nodes of ani-mals with chronic diarrhea (23.3% � 4.0%, Table 2). However,the trend (P � 0.05) for an increased % of activated lympho-cyte populations in animals with chronic diarrhea was observedin both the mesenteric lymph node and the spleen. When theCD4� and CD8� populations were compared, a larger pro-portion of activated cells (CD69�) and a lower proportion ofnaive cells (CD45RA�) were observed in the CD4� T-cellpopulation than in the CD8� T-cell population in both groupsof animals (Table 2).

Cytokine gene expression. Statistically significant upregula-tion (P 0.05) of cytokine genes specific for IL-1�, IL-3, andTNF-� in both the mesenteric lymph nodes and the spleens ofanimals with chronic diarrhea versus clinically healthy animals(Fig. 4) was detected. Each of these cytokine genes showed atleast a 2.5-fold increase in animals with chronic diarrhea com-pared to those without diarrhea. The level of expression ofthese cytokine genes in normal animals was 20 to 30% of theinternal standard control (�-actin), whereas in animals withchronic diarrhea these cytokine genes were expressed at 60 to70% of the internal standard control. IL-10 and IL-16 geneexpression in both the mesenteric lymph nodes and the spleenwas not significantly different between the two groups (Fig. 4).The remainder of the 18 tested cytokine genes were expressedat very low or undetectable levels.

FIG. 2. Percentages of infectious agents detected from stools of 100 rhesus macaques either with (�, n 50) or without (■ , n 50) symptomsof chronic diarrhea. A statistically significant difference (P 0.05) between the two groups is indicated by an asterisk.

4082 SESTAK ET AL. INFECT. IMMUN.

DISCUSSION

The gastrointestinal immune system has recently been im-plicated as important in the pathogenesis of numerous diseasesand syndromes, including AIDS (39, 40), and of IBD disorderssuch as ulcerative colitis and Crohn’s disease (34). A healthygastrointestinal tract is critical for research studies involvingpathogenesis, as well as novel mucosal vaccine and gene ther-apy approaches utilizing genetically modified viruses, bacteria,or synthetic delivery systems. Intestinal malabsorption, chronicdiarrhea, and wasting are frequent manifestations of humanimmunodeficiency virus infection and, in many cases, the pre-senting illness (17, 21, 30). Similar findings have been observedin the SIV/macaque model of AIDS (18, 35). In SIV-infectedmacaques, large numbers of infected lymphocytes are foundthroughout the intestinal lamina propria within days of intra-venous virus inoculation (18, 29, 38). Many of the pathologicalchanges, such as the rapid loss of intestinal CD4� T lympho-cytes, are clearly due to SIV infection. However, other alter-ations, such as the morphological changes and persistent diar-rhea, may be associated with opportunistic infections oralteration in intestinal flora. Developing strategies to preventor treat these enteric infections and elucidate their role, or lackthereof, in AIDS enteropathy and disease progression is thefocus of numerous studies (a search of the CRISP database forthe terms opportunistic infection and AIDS returned 469 hitsfor the year 2001). Many of these studies are hampered by thefact that most captive macaques in existing colonies have beenexposed to these agents. Thus, our study sought to identify theenteric pathogens most frequently associated with chronic di-arrhea in normal, immunocompetent rhesus macaques and toelucidate their impact on the occurrence of chronic enteroco-litis and the host immune system in general.

We identified the presence of gastrointestinal pathogenssuch as Campylobacter spp., Shigella flexneri, Yersinia enteroco-litica, adenovirus, and Strongyloides fulleborni in rhesus ma-caques with chronic diarrhea. These organisms are known tobe associated with chronic enterocolitis not only in nonhumanprimates but also in humans (5, 8, 20, 22, 36, 37). A wide rangeof histopathological changes in intestinal tissues collected fromanimals with chronic diarrhea reflected the presence of a num-

ber of enteric pathogens and their cumulative effect. It is likelythat additional agents beyond those identified in the presentstudy were also contributing to the chronic enterocolitis. How-ever, the detection of microorganisms such as Mycobacteriumspp. (Mycobacterium avium and Mycobacterium simiae), theCampylobacter-like bacterium Helicobacter cinaedi, Brachyspirasp., or cytomegalovirus would require more focused and spe-cialized examination (15, 19).

Although the opportunistic microsporidium Enterocytozoonbieneusi was more prevalent in animals with chronic diarrhea inthe present study than in clinically healthy animals, it was notsignificantly associated with the onset of diarrhea. This is con-sistent with our earlier findings. We previously reported thatthere is a significant increase in Enterocytozoon bieneusi shed-ding only in individuals with very low levels of peripheralCD4� T lymphocytes (33).

Rotaviruses were not detected in the present study. Com-pared to human infants, maximum shedding of rotavirus anti-gens in feces of rhesus monkeys is expected at the age ofapproximately 4 months, a time when passive, maternally ac-quired immunity subsides. In our study, the age of monkeyswas 6.5 months and older, and these animals were showingsymptoms of chronic diarrhea; therefore, we would not haveexpected to detect rotavirus in association with their disease.However, exposure to rotavirus will occur in most convention-ally reared rhesus macaques at very young ages when it is likelyto result in infection that produces lower levels of shedding andmild disease. In the majority of adult rhesus macaques thatpossess actively acquired rotavirus immunity, reinfections oc-cur only subclinically or the disease is very mild, with little virusshedding in the feces (unpublished results).

Detection of adenoviruses in our study was based on the useof a commercial polyclonal reagent that is capable of detectingthe presence of any of the 49 known adenovirus serotypes(Dako), including those reported to cause gastroenteritis inhumans (serotypes 40 and 41) and monkeys (serotypes 17, 20,and 32) (36, 37). Adenoviruses were associated in our studywith chronic diarrhea.

EPEC strains were previously recognized as opportunisticpathogens in SIV-infected rhesus macaques (22). Among rhe-sus macaques with AIDS, EPEC was identified in 28% ofanimals (22). Moreover, in 7.3% of animals dying with AIDS,EPEC was the sole pathogen detected in the gastrointestinaltract (22). In our study, the Escherichia coli strains that wereidentified as carriers of enteric virulence genes were found inup to 25% of animals regardless of clinical symptoms of diar-rhea. It is important to note, however, that among our immu-nocompetent animals, of the seven Stx2c-virulence gene-car-rying Escherichia coli isolates only two actively produced Verocell toxins. The fact that 32% of clinically healthy animals hadsome past history of clinical diarrhea might explain the detec-tion of several gastrointestinal pathogens from this “clinicallyhealthy” category.

We attempted to elucidate what differences, if any, would beseen with respect to activated and/or naive CD4� and CD8�

lymphocytes in gut-associated inductive lymphoid tissues andsystemic inductive lymphoid tissues from animals with or with-out chronic enterocolitis. Immunological involvement of bothmajor T-lymphocyte populations (CD4� and CD8�) in ani-mals with chronic diarrhea was indicated by the increased

TABLE 2. Activated-T-cell phenotypesa

Antigen expression

Mean % activated T cells � SD in:

Mesenteric lymph nodes Spleens

Clinicallyhealthyanimals

Animalswith

chronicdiarrhea

Clinicallyhealthyanimals

Animalswith

chronicdiarrhea

CD4�

CD45RA� CD69� 48.5 � 8.2 24.1 � 7.1 42.3 � 2.8 24.7 � 7.3CD45RA� CD69� 12.3 � 4.0 23.3 � 4.0b 12.6 � 4.3 24.0 � 9.8CD45RA� CD69� 5.1 � 0.7 5.2 � 2.4 5.9 � 1.7 8.9 � 3.7

CD8�

CD45RA� CD69� 77.7 � 8.9 50.7 � 4.4 52.6 � 9.3 33.5 � 9.9CD45RA� CD69� 4.7 � 2.6 13.9 � 5.3 11.3 � 5.2 10.8 � 5.9CD45RA� CD69� 5.6 � 1.0 9.8 � 4.2 19.3 � 2.8 43.5 � 10.9

a Values represent subsets of animals with chronic diarrhea (n 3) andclinically healthy controls (n 3).

b A statistically significant difference (P 0.05) between the two groups isindicated.

VOL. 71, 2003 CHRONIC ENTEROCOLITIS IN CAPTIVE RHESUS MACAQUES 4083

presence of activated (CD69�) cells, although a significantincrease was measured only in the CD4� T lymphocytes iso-lated from mesenteric lymph nodes. This increased presence ofactivated T lymphocytes with T helper cell characteristics mostlikely reflected the presence of multiple enteric pathogens overa long period of time, possibly associated with ligand reactiv-ities to multiple antigens. In patients with IBD, typically withchronic inflammation of the terminal ileum and colon, CD4� Tlymphocytes, cross-reactive with different species of aerobicand anaerobic bacteria, have been detected (12). Although the

precise etiology of IBD is not clear, it is speculated that hy-perresponsive T lymphocytes in IBD patients react with com-mensal bacterial microflora of the gut and cause chronic in-flammation (12). In the present study the presence of multipleenteric pathogens in rhesus macaques was associated withchronic inflammation of the colon and an increased percentageof activated (namely, CD4�) T lymphocytes in gut-associatedand systemic lymphoid tissues.

Consistent with the presence of inflammation and the in-creased percentage of activated cells, we observed upregula-

FIG. 3. Flow cytometry histograms of mononuclear cells isolated from lymphoid tissues of an animal with chronic diarrhea (J079) and aclinically healthy control (T080). Gating was performed on population of bright CD3� T lymphocytes as determined by a single-color-stainedsample. Major differences between the two animals can be seen between the percentages of activated (CD69) and naive (CD45RA�) CD4� andCD8� lymphocyte subsets in both mesenteric lymph nodes and spleens.

4084 SESTAK ET AL. INFECT. IMMUN.

tion of IL-1-�, IL-3, and TNF-� cytokine genes. This finding isin accord with studies conducted with murine and rhesus mod-els of human chronic colitis wherein increased expression ofIL-1 and TNF-� was related to the development of intestinalpathology (4, 26, 41). Chronic transmural colitis in STAT4transgenic mice was characterized by the infiltration of CD4�

T lymphocytes secreting TNF and IFN-� (41). In vitro-culturedexplants of a rhesus colon with conditions of colitis resulted inincreased production of IL-1 and TNF-� in contrast to culturesderived from a normal colon (26). It was suggested that inhi-bition of inducible nitric oxide synthase (iNOS) might abrogatethe severity of chronic colitis (26). However, the complexity offactors that may affect IBD, such as cytokine network interac-tions with intestinal vascular endothelium and/or neurons thatare known to constitutively express iNOS, still needs to be fullyelucidated. The recent focus of research on the development of

novel strategies for treating human patients with IBD hasshifted to immune therapies. Trials with Crohn’s disease pa-tients treated with anti-TNF-� antibodies demonstrated dra-matic improvement, as scored by colonoscopy findings (2, 3,11).

The increased immunological reactivities (i.e., the presenceof CD69� T lymphocytes and the upregulation of IL-1�, IL-3,and TNF-� cytokine genes) were detected in the present studynot only in gut-associated lymphoid tissues but also in systemiclymphoid tissues represented by the spleen. This suggests thatthe prevention of chronic enterocolitis in nonhuman primatesis critical in order to increase the usefulness of this animalmodel for studies involving vaccine or gene therapy research.On the other hand, the rhesus colitis model could also beexplored as an in vivo model in trials with new drugs andimmune regimens that are aimed to reduce or abrogate IBD.

FIG. 4. The relative abundance of mRNA transcripts specific for IL-1�, IL-3, IL-10, IL-16, and TNF-� cytokine genes is shown and comparedbetween the animals with chronic diarrhea (p, n 3) and clinically healthy controls ( , n 3) in mesenteric lymph nodes (A) and spleens (B).The means � the standard deviations are shown for cytokine genes that were found to be significantly different (a P value of 0.05 is indicatedby an asterisk for IL-1�, IL-3, and TNF-�), along with the two genes (IL-10 and IL-16), for which no significant difference was observed. The IL-1�,IL-3, and TNF-� gene expression in mesenteric lymph nodes was significantly higher in animals with chronic diarrhea (P 0.02, 0.03, and 0.02,respectively). Similarly, IL-1�, IL-3, and TNF-� gene expression in the spleen was significantly higher in animals with chronic diarrhea at P 0.05,0.03, and 0.05, respectively.

VOL. 71, 2003 CHRONIC ENTEROCOLITIS IN CAPTIVE RHESUS MACAQUES 4085

ACKNOWLEDGMENTS

This study was supported by Public Health Service grant DK50550.Partial support was provided by TNPRC base grant RR00164.

The technical assistance of Ayanna Jefferson, Ann Bennett, LisaBowers, Maurice Duplantis, and Kevin Callahan is greatly appreciated.We thank Gary B. Baskin and John D. Clements for careful review andsuggestions regarding the manuscript.

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Editor: A. D. O’Brien

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