Pathophysiology of the Rhesus Macaque Model for ... · Lisa N. Henning, Stephen M. Miller, Dennis H. Pak, Amber Lindsay, David A. Fisher, Roy E. Barnewall, Crystal M. Briscoe, Michael

Pathophysiology of the Rhesus Macaque Model forInhalational Brucellosis

Lisa N. Henning, Stephen M. Miller, Dennis H. Pak, Amber Lindsay, David A. Fisher, Roy E. Barnewall, Crystal M. Briscoe,Michael S. Anderson, and Richard L. Warren

Battelle, Columbus, Ohio, USA

The objective of this study was to characterize the rhesus macaque (RM) as a model for inhalational brucellosis in support of theU.S. Food and Drug Administration’s (FDA) Animal Rule. The pathophysiology of chronic Brucella melitensis aerosol infectionwas monitored in two phases that each occurred over an 8-week time period; dose escalation (8 RMs; targeted doses of 5.0E�03,5.0E�04, or 5.0E�05 CFU/animal or the unchallenged control) and natural history (12 RMs; targeted dose of 2.50E�05 CFU/animal or the unchallenged control). RMs given an aerosol challenge with B. melitensis developed undulating fevers (6/6 phase I;8/9 phase II), positive enriched blood cultures (5/10; phase II), and bacterial burdens in tissues starting 14 to 21 days postchal-lenge (6/6 phase I; 10/10 phase II). In addition, 80% (8/10; phase II) of infected RMs seroconverted 14 to 21 days postchallenge.RMs developed elevations in certain liver enzymes and had an increased inflammatory response by 3 weeks postchallenge asshown by increases in C-reactive protein (6/8) and neopterin (4/8), which correlated with the onset of a fever. As early as 14 dayspostchallenge, positive liver biopsy specimens were detected (2/8), and ultrasound imaging showed the development of spleno-megaly. Finally, histopathologic examination found lesions attributed to Brucella infection in the liver, kidney, lung, and/orspleen of all animals. The disease progression observed with the RMs in this study is analogous to human brucellosis pathophys-iology. Thus, the results from this study support the use of the RM as an animal model for inhalational brucellosis to evaluate theefficacy of novel vaccines and therapeutics against B. melitensis.

Brucella is a Gram-negative coccobacillus that includes certainspecies that can cause disease in animals and humans. Brucella

melitensis, one of the major species of Brucella, is an etiologic agentfor brucellosis. This highly infectious pathogen can infect animalsand humans, and it is considered to be one of the most severehuman pathogens in the Brucella genus (25). Humans are usuallyinfected following contact with infected animals or ingestion ofcontaminated milk, milk products, and meat. In addition, brucel-losis is one of the most common laboratory-acquired infections,as well as the most common zoonosis (11). While B. melitensis isnot typically considered a lethal pathogen in humans (less than5% fatality rate), it can cause significant disease. Human brucel-losis presents as a prolonged febrile illness with flu-like symptoms,such as night sweats, headache, depression, and arthritis. Chronicillness can lead to meningitis and endocarditis. In addition, re-lapses can occur, even with antibiotic treatment (13). Notably,humans can remain asymptomatic for weeks, months, or evenyears while infected with this pathogen (3).

Brucella species are considered potential biological warfareagents due to their high infectivity, ability to debilitate infectedpeople, and the persistent nature of human disease. In addition,Brucella is easily disseminated by aerosols and can induce disease(20). Therefore, it is a priority to develop and use appropriateanimal models to evaluate the efficacy of novel vaccines and ther-apeutics against inhalational brucellosis.

Nonhuman primates (NHPs) have been used as a model forseveral diseases due to their similar susceptibilities and diseaseprogression as those seen in humans, including brucellosis infec-tion (7–9, 12, 15, 24). While there are only minimal data fromNHP studies, one study showed that rhesus macaques (RMs) weresusceptible to infection by aerosolized B. melitensis as demon-strated by tissue burden and histopathology (18). A second studymonitored the tissue burden and histopathology of RMs for 7 days

after aerosol challenge with a high dose of Brucella suis (24). Awell-understood pathophysiological mechanism of toxicity and asufficiently well-characterized animal model for predicting theresponse in humans are two key components of the Animal Rule(21 CFR 314.600 for drugs and 21 CFR 601.90 for biological prod-ucts). Therefore, the current study was designed to thoroughlyevaluate the pathophysiology of B. melitensis 16 M by aerosol in-halation in RMs over an 8-week time period, in order to develop awell-characterized RM model of brucellosis. The first phase of thisstudy was conducted to determine an optimal target challengedose for assessing the pathophysiological course of infectionduring an 8-week time period. The clinical parameters examinedincluded telemetry (temperature, activity, electrocardiogram[ECG], and cardiovascular function), clinical observations, bloodculture, body weights, and tissue burden. In addition, liver biopsysamples and splenomegaly were assessed using ultrasound imag-ing. Gross necropsies were performed at the scheduled euthanasiatime point, and tissues were collected and histopathology evalu-ated by a veterinary pathologist.

In the second phase of this study, aerosol-challenged animalsreceived a targeted dose of 250,000 CFU of B. melitensis 16 M.Animals were serially euthanized over an 8-week postchallengeperiod so that the natural history of brucellosis in RMs could be

Received 29 August 2011 Returned for modification 23 September 2011Accepted 22 October 2011

Published ahead of print 7 November 2011

Editor: S. R. Blanke

Address correspondence to Lisa N. Henning, [email protected].

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.05878-11

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characterized. In addition to the parameters assessed in the firstphase of this study, the second phase of this study evaluated clin-ical hematology and clinical pathology, C-reactive protein (CRP),and several innate and immune markers during the 8-week post-challenge period. The information obtained from this study willbe used to further expand the RM model for future evaluation ofvaccine and therapeutic efficacies against aerosol exposure to B.melitensis.

MATERIALS AND METHODSAnimals. A total of 20 RMs (Macaca mulatta) were used for the twophases of this study (Covance; Alice, TX). All animals were more than 2years old and greater than 3 kg. Each animal was implanted with a telem-etry unit (D70-PCTP; Data Sciences International, St. Paul, MN) approx-imately 4 weeks prior to aerosol challenge. All animals were negative forsimian immunodeficiency virus (SIV), simian T-lymphotrophic virus 1(STLV-1), cercopithecine herpesvirus 1 (herpes B virus), and for simianretrovirus 1 (SRV1) and SRV2 by PCR. All RMs were in good health, freeof malformations, and exhibited no signs of clinical disease. The animalprotocol was approved by Battelle’s Institutional Animal Care and UseCommittee (IACUC) and the Department of Defense Animal Care andUse Review Office (ACURO).

The first phase of this study used eight male RMs, and the second phaseused 12 RMs (50% females, 50% males). These animals were randomizedby weight into one of four groups (phase I) or one of six groups (phase II)by using the SAS statistical software utilizing the Plan procedure. Thesample sizes of two animals per group constituted the minimum numberfor which any comparison could be made for a difference in study param-eters (i.e., hematology, clinical chemistry, blood culture, and tissue bur-den) between aerosol challenge groups and time points. The selection offive aerosol challenge groups (aside from controls) in phase II providedthe requisite level of detail for characterizing the time course variabilitiesin hematology, clinical chemistry, and bacterial burden parameters.

Preparation of challenge material. B. melitensis 16 M biovar 1 ATCC23456 (Super Master Cell Bank [SMCB]) was obtained from the Ameri-can Type Culture Collection (ATCC; Manassas, VA; original preparation,1998), and it was used to prepare several vials of a master cell bank (MCB)by suspending fresh colonies from the SMCB plate culture in brucellabroth (BB) plus approximately 20% glycerol. A vial of the MCB was platedfor isolation and incubated at 37°C for 67 h on blood agar. The streak platecultures were examined for purity and colony morphology, and a Gramstain was performed on an isolated colony. A flask culture inoculum sus-pension was then prepared by suspending freshly isolated colonies in ap-proximately 6 ml of BB to an optical density at 600 nm (OD600) of 0.192.This suspension was used to inoculate a 1-liter baffled flask with a ventedcap containing 100 ml of sterile BB with 3.70E�08 B. melitensis CFU. The1-liter flask culture was incubated at 37°C (shaking at 250 rpm) for ap-proximately 24 h. After incubation, a Gram stain was performed on theflask culture material to confirm purity. To make the working cell bank(WCB) material, 25 ml of sterile 80% glycerol was added to 75 ml of thebroth culture for a final glycerol concentration of 20%. This WCB wasdispensed into sterile vials and frozen at or below �70°C. Several daysafter preparation, randomly chosen vials were thawed, enumerated, andcharacterized to ensure the identity and purity of the WCB, which wasused for all challenges.

For each aerosol challenge day, a fresh B. melitensis suspension wasprepared from a new, single-use vial of the WCB, using aseptic technique.Briefly, two modified Thayer Martin (MTM) agar plates were streakedwith a thawed sample of the WCB for isolation and incubated at 37°C forapproximately 72 h (phase I) or 96 h (phase II). For each phase, twoovernight broth culture flasks were prepared (one for challenge prepara-tion and one for backup) by suspending fresh colonies from the platecultures in �6 ml of sterile BB, which were then used to inoculate 100 mlof sterile BB in 1-liter baffled flasks with vented caps. Overnight cultureswere incubated at 37°C (shaking at 250 rpm) for approximately 24 h for

each phase. The OD600 of the culture used to prepare the challenge mate-rial was 0.563 (phase I) or 0.444 (phase II). These cells were pelleted at10,000 relative centrifugal force for 10 min at 26°C. The pellet was thenwashed by resuspension in sterile phosphate-buffered saline containing0.01% (wt/vol) gelatin and 9.7% (wt/vol) trehalose (BSGT). This suspen-sion was again centrifuged, followed by suspension in BSGT to a targetOD600 of 1.5, with an anticipated concentration of 5.81E�09 CFU/ml.The adjusted suspension was then diluted in BSGT to the target startingnebulizer concentration(s) and stored on ice until dissemination. Ap-proximately 8 ml of this challenge material was placed into the nebulizerfor each aerosol run.

Aerosol challenge. A head-only aerosol exposure system was utilizedto deliver target B. melitensis 16 M aerosol doses. The aerosol exposuresystem is capable of exposing one NHP at a time. Exposures were per-formed as previously described (23). The target challenge doses for phaseI were as follows: 5.00E�03 CFU/animal (group 1), 5.00E�04 CFU/ani-mal (group 2), and 5.00E�05 CFU/animal (group 3). Group 4 was un-challenged control animals. The target challenge dose to assess chronicinfection in phase II was 2.50E�05 CFU/animal (groups 5 to 9), whichwas based upon tissue burden and body temperature data from phase I.Group 10 was unchallenged control animals for phase II.

Clinical evaluation. Prior to placement on study, all RMs were im-planted with D70-PCTP telemetry transmitters (Data Sciences Interna-tional, St. Paul, MN) using aseptic surgical techniques as previously de-scribed (23). Briefly, the RMs were lightly anesthetized by administrationof ketamine (20 mg/kg of body weight) followed by balanced gaseousanesthesia (approximately 100% oxygen and 1 to 3% isoflurane) to main-tain surgical anesthesia. The telemetry transmitter was inserted into theabdomen and attached to the wall of the abdominal cavity. The cardiacpressure catheter was inserted into the femoral artery and advanced intothe iliac artery, where it was secured in place. The pleural pressure catheterwas implanted beneath the serosal layer of the esophagus within the tho-racic cavity (used to measure respiratory pressure). The ECG leads wereplaced against the abdominal muscle along the rib cage below the apex ofthe heart and against the thoracic wall. All RMs had recovery periods ofapproximately 4 weeks prior to challenge and were in good health at thetime of challenge.

Baseline body temperature, activity, and cardiovascular function(heart rate, systolic/diastolic pressure, pulse pressure, mean pressure, andrespiratory rate) were collected by the implanted telemetry unit beginning4 days prechallenge. Data were collected for 30 s every 15 min for theduration of the postchallenge period (approximately 8 weeks). The fol-lowing parameters were collected throughout the monitoring period:temperature, activity, blood pressure, heart rate, pulse pressure, respira-tion rates and pressures, and ECG.

Animals were observed twice daily prechallenge and postchallengeuntil the end of in-life. Any observed clinical signs (e.g., lethargy, loss ofappetite, labored breathing) were documented.

Baseline body weights were obtained prior to the challenge day and onthe day of challenge. Postchallenge body weights were measured weekly.

Blood samples were collected from a femoral artery or vein and processedfor clinical chemistry and hematology analysis. Whole blood was collected inEDTA tubes for bacterial culture. Serum was isolated from whole blood col-lected in serum separator (SST) tubes and used for serology and determina-tion of inflammatory biomarker (neopterin) concentrations by enzyme-linked immunosorbent assay (ELISA). Blood samples for clinical chemistry,CRP, hematology, and serological and inflammatory biomarkers were drawn5 days prior to challenge and on days 7, 14, 21, 28, 35, 42, and 56 postchal-lenge. Blood samples for bacteriological culture were obtained 5 days prior tochallenge and on the day of scheduled euthanasia.

Blood was collected for hematology (Siemens Advia 120 hematologyanalyzer), CRP, and clinical chemistry evaluation (Siemens Advia 1200chemistry analyzer), which included white blood cell count (WBC), dif-ferential leukocyte count (percentage and absolute), neutrophil/lympho-cyte ratio (N/L ratio), hemoglobin (HGB), hematocrit (HCT), red blood

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cell count (RBC), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), mean corpuscular hemoglobin concentration(MCHC), red cell distribution width (RDW), platelet count (PLT), andmean platelet volume (MPV). Clinical chemistry evaluation included al-anine aminotransferase (ALT), aspartate aminotransferase (AST), alka-line phosphatase (ALP), gamma-glutamyl transferase (GGT), lactate de-hydrogenase (LDH), sorbitol dehydrogenase (SDH), total bilirubin, totalprotein, albumin, globulin, albumin/globulin (A/G) ratio, blood urea ni-trogen (BUN), creatinine, BUN/creatinine ratio, glucose, sodium, potas-sium, chloride, calcium, and phosphorous. CRP analysis was performedon serum collected from whole-blood samples. Normal clinical ranges forclinical chemistry and hematology values for RMs were provided by Sie-mens Advia.

Whole blood was collected, and approximately 30 to 40 �l of bloodwas used to test for bacteremia by culture for samples in the first phase.Briefly, each whole-blood sample was streaked onto MTM plates andincubated at 37°C for approximately 96 h. One hundred microliters ofwhole blood from each animal in the second phase of the study was en-riched in 3 ml of brucella broth selectively supplemented with modifiedbrucella selective supplement (MBSS; containing final concentrations of15 IU polymyxin B, 75 IU bacitracin, 0.3 mg cycloheximide, 15 �g nali-dixic acid, 300 IU nystatin, and 60 �g vancomycin [Oxoid Limited, Cam-bridge, United Kingdom]) for approximately 96 h at 37°C prior to plating30 to 40 �l of the sample on MTM agar. Plates were incubated for approx-imately 72 h at 37°C and were examined for the presence or absence of B.melitensis 16 M (limit of detection [LOD], 25 CFU/ml).

A portion of the serum isolated from whole blood was used to assessbiomarker kinetics, which included a microagglutination assay, anti-Brucella IgG, anti-Brucella IgM, and neopterin ELISAs. Prior to testing theRM serum samples by microagglutination, the optimal antigen dilutionwas first determined by checkerboard titration as described by Gaultneyet al. (14). The microagglutination test was performed according to themethod reported by Bettelheim et al. (2), with the following modification:Rose Bengal antigen was used instead of milk ring test antigen, with noadditional bovine serum added to the wells. The absence of agglutinationwas indicated by a running button of red-stained Brucella bacteria. Agglu-tination was indicated by the complete dispersion of antigen or by a di-minished discrete nonrunning button of red-stained bacteria remainingin the center of the well. The highest dilution with an agglutination pat-tern was considered the microagglutination titer. Samples were run intriplicate, and the average value is reported. A 1:2,500 dilution was thehighest dilution tested.

Anti-Brucella IgG and IgM levels were determined using ELISA kits(catalog number RE56841 for IgG and RE56821 for IgMl; IBL Interna-tional). Assays were performed according to manufacturer recommenda-tions. The neopterin ELISA (catalog number RE59321; IBL International)was performed according to the manufacturer recommendations. In ad-dition to the CRP analysis with the Advia system, an ELISA for measuringCRP was performed with a commercial kit (catalog number 2210-4LD51071; Life Diagnostics). This assay was performed according to man-ufacturer recommendations. All ELISA samples were run in triplicate, andthe average values are reported.

Ultrasound-guided biopsies of the liver were conducted weekly bya veterinarian on each RM. Animals were anesthetized with 3 to 6mg/kg tiletamine (Telazol, intramuscularly [i.m.]) prior to the proce-dure. Samples were collected with a 22- to 23-gauge microfine needle,and animals were then treated with 0.01 to 0.02 mg/kg buprenorphine(Buprenex, i.m.). The liver biopsy sample obtained was enriched inbrucella broth supplemented with MBSS for approximately 96 h at37°C. Following the enrichment, 30 to 40 �l of the sample was platedon MTM agar, incubated for approximately 72 h at 37°C, and thenanalyzed qualitatively for bacteria.

In addition, during each ultrasound-guided biopsy procedure, the vet-erinarian examined ultrasound images of the spleen for splenomegaly.The size of the spleen for each animal was measured from the sonogram

images and recorded weekly during the second phase of the study (mini-abdominal probe; 6.6 mHz at 70% gain).

During necropsy, samples of the spleen, liver, kidney, and lung werecollected, weighed, and placed in a known volume of sterile phosphate-buffered saline plus 0.01% (wt/vol) gelatin (BSG). The tissues were ho-mogenized, and serial dilutions were plated on MTM agar. After 72 to 96h of incubation at 37°C, colonies on the plates were counted. The limit ofquantitation (LOQ) was equal to 100 CFU/g, and the LOD was equal to 3CFU/g.

Necropsy and histopathology. Necropsies were performed on allNHPs with tissues collected and examined by the study pathologist.Lungs, liver, kidney, testes/ovaries, spleen, brain, and lymph node tissueswere harvested and formalin fixed. In addition, in the second phase of thestudy, the knee joint was collected and examined for Brucella-inducedarthritis and bone marrow isolated from the sternum was harvested tolook for evidence of Brucella-induced lesions, since lesions have been de-tected in the bone marrow of human brucellosis patients (22). The histo-pathology of all tissues was characterized by a board-certified veterinarypathologist. All microscopic findings were graded semiquantitatively ac-cording to the following scale, with the associated numerical score used tocalculate average severity grades for each lesion by group. Minimal (grade1) represented the least detectable lesion; mild (grade 2) represented aneasily discernible lesion; moderate (grade 3) represented a change affect-ing a large area of the represented tissue; marked (grade 4) represented alesion that approached maximal.

Statistical methods. Animal group randomizations were performedusing SAS version 9.1 (SAS Institute, Cary, NC). The telemetry data foreach animal in the postchallenge period were adjusted to subtract theanimal’s average response at the same time of day in the prechallengeperiod to provide baseline-adjusted values. Baseline-adjusted postchal-lenge 1-h averages were calculated for each animal as follows: 24-houraverages were calculated for the prechallenge baseline data for each ani-mal. Then, for each animal, the baseline-adjusted postchallenge value wascalculated by subtracting the baseline average value from each postchal-lenge value at that hour. Finally, baseline-adjusted, postchallenge 1-h av-erages were calculated from the four 15-min readings in each postchal-lenge hour to obtain hourly baseline-adjusted postchallenge averagevalues. Mean telemetry data were graphed over 8-h intervals for chal-lenged and also unchallenged animals that were not euthanized until day56 (groups 9 and 10) and could therefore be used to observe the kinetics ofthese parameters.

RESULTSAerosol challenge. The RMs were aerosol challenged with B.melitensis 16 M bv. 1. For the first phase of this study, the averageaerosol exposure dose for animals challenged was 5.77E�03 CFU,3.60E�04 CFU, and 4.90E�05 CFU B. melitensis 16 M for groups1 to 3, respectively. The mass median aerodynamic diameter(MMAD) of aerosolized B. melitensis ranged from 2.63 to 2.70�m, which is consistent with a particle size that will deposit withinthe lower respiratory tract.

For the second phase of this study, the average aerosol chal-lenge dose was 3.76E�05 B. melitensis 16 M CFU per animal, witha range of 3.07E�05 to 4.47E�05 B. melitensis 16 M CFU (Table1). The MMAD of aerosolized B. melitensis was 2.81 �m, which isconsistent with a particle size that will deposit within the lowerrespiratory tract.

Body weights. There was little to no change in body weights ofanimals that were challenged with B. melitensis compared to un-challenged animals. However, one animal in group 9 (a phase IIfemale, scheduled euthanasia on day 56 postchallenge) lost 15%body weight between days 21 and 42 postchallenge. This animalstarted to gain weight by day 49 postchallenge.

Clinical observations. Clinical observations were performed

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twice daily throughout the postchallenge period. All of the ani-mals in the first phase of the study were normal throughout thepostchallenge period, except for an occasional stool abnormality(i.e., soft stool) or decreased appetite, similar to the unchallengedanimals. All animals except for one animal (group 9 female, sched-uled euthanasia on day 56 postchallenge) in the second phase werenormal throughout the postchallenge period, except for an occa-sional stool abnormality. Two animals vomited after challenge butwere normal for the remainder of the study. From days 27 to 30postchallenge, the group 9 animal was observed to be weak. How-ever, this animal was observed to be normal after day 30 throughthe end of the in-life period.

Telemetry. While several parameters were assessed by teleme-try, changes were most apparent for body temperature and activityin challenged animals compared to unchallenged animals. Figure1 shows the average kinetics of nine telemetry parameters for twoanimals challenged with the targeted dose of 2.50E�05 CFU B.melitensis and for two unchallenged animals that were monitoreduntil day 56 postchallenge (phase II animals; groups 9 and 10).Body temperature increased and activity decreased by 3 weekspostchallenge for the challenged animals, while body temperatureremained steady and activity increased for the unchallenged ani-mals during this time period. Notably, animals still maintainedthe diurnal rhythm pattern while exhibiting a fever (greater than40°C). The body temperature for the challenged animals started todecline to closer to baseline by day 42 postchallenge while stillexhibiting some small fluctuations.

The expiratory and inspiratory times, heart rate, respiratoryrate, and systolic and diastolic pressure patterns were similar for

the challenged and unchallenged animals during the 56-day mon-itoring period. However, blood pressure parameters appeared tobe affected by brucellosis infection in phase I (data not shown).The phase I average baseline-adjusted maximum value for dia-stolic blood pressure for groups 1 to 3 (targeted challenge dosage,5.00E�03 CFU, 5.00E�04 CFU, or 5.00E�05 CFU, respectively)was 2.5 to 5 times greater than the average baseline-adjusted max-imum value for the unchallenged control (group 4) animals, andthe average baseline-adjusted maximum value for systolic bloodpressure for groups 1 to 3 was 1.7 to 4.2 times greater than theaverage baseline-adjusted maximum value for the unchallengedcontrol (group 4) animals. The phase II results, however, did notshow an effect of brucellosis infection on blood pressure parame-ters, as the baseline-adjusted maximum values for the challengedanimals were similar to those for the unchallenged control ani-mals. The lack of a difference between the challenged and controlanimals in phase II was at least partially due to the euthanasiaof animals in groups 5 to 7 (euthanized on days 14, 21, or 28,respectively) on or before day 28 postchallenge.

It appeared that the pulse pressure for the unchallenged ani-mals decreased during the postchallenge period, but this was dueto a higher baseline pulse pressure average for this group, whichresulted in decreased baseline-adjusted values during the moni-toring period (Fig. 1).

Blood samples for bacterial culture. All animals from phase Iwere negative by blood culture on the last day of in-life, but all ofthese animals were positive for B. melitensis 16 M in the lung, liver,and/or spleen tissue. The discrepancy between blood culture andtissue burden could have been due to the concentration of bacteriain the blood being below the LOD. Thus, detection from directplating may have been unlikely due to the limited volume of bloodplated. To improve the probability of detecting low levels of bac-teria in whole blood, enrichment culture techniques were usedprior to plating for bacterial identification for phase II. Unlike thephase I results, 50% (5/10) of the challenged animals in phase IIwere positive by culture for B. melitensis 16 M at the scheduledeuthanasia time points. Both animals were positive on day 14 ateuthanasia (2/2) and on day 28 at euthanasia (2/2), and one ani-mal was positive on day 42 at euthanasia (1/2). Both challengedanimals euthanized on day 56 were blood culture negative andtissue burden positive (bacteria levels below the LOQ).

Clinical pathology and C-reactive protein. In humans, bru-cellosis causes a mild elevation in liver enzymes and an increase inCRP levels (3, 5, 10). In this study, we observed that RMs hadincreases in liver enzymes AST, CRP, ALT, ALP, and LDH duringthe 8-week postchallenge period. Other clinical chemistry param-eters (i.e., creatinine, BUN, and glucose) in infected RMs were notdifferent from baseline, which is similar to human brucellosis(24). Figure 2 illustrates the trends for some of the clinical pathol-ogy parameters during the 8-week postchallenge period for chal-lenged and unchallenged animals that were not euthanized untilday 56 (groups 9 and 10) and could therefore be used to show thekinetics of these parameters.

AST levels remained within the clinically normal range at alltime points, except for one challenged animal on day 28. The ASTlevels for animals challenged with B. melitensis 16 M were onlyslightly above the levels for unchallenged animals from day 21 today 56 postchallenge.

All animals challenged with B. melitensis had increased levels ofCRP by day 21 postchallenge compared to baseline, while CRP

TABLE 1 Summary of challenge data

Phase andgroup

Day ofscheduledeuthanasia(postchallenge) Sex

Age(yrs)

Weight(kg)a

Targetchallengedose(CFU/RM)

Estimatedinhaleddose(CFU)b

Phase I1 56 M 4 4.3 5.00E�03 5.44E�031 56 M 4 4.4 5.00E�03 6.10E�032 56 M 4 4.3 5.00E�04 3.18E�042 56 M 4 5.0 5.00E�04 4.02E�043 56 M 4 4.2 5.00E�05 4.68E�053 56 M 4 4.9 5.00E�05 5.11E�054 56 M 4 4.6 0.00E�00 0.00E�004 56 M 4 5.5 0.00E�00 0.00E�00

Phase II5 14 F 4 5.0 2.50E�05 3.24E�055 14 M 2 3.7 2.50E�05 3.17E�056 21 F 4 5.5 2.50E�05 4.39E�056 21 M 3 3.6 2.50E�05 3.07E�057 28 F 2 3.6 2.50E�05 4.30E�057 28 M 2 3.8 2.50E�05 3.48E�058 42 F 2 3.7 2.50E�05 4.47E�058 42 M 2 3.7 2.50E�05 4.14E�059 56 F 3 6.4 2.50E�05 4.04E�059 56 M 3 3.9 2.50E�05 3.39E�0510 56 F 2 4.3 0.00E�00 0.00E�0010 56 M 3 3.7 0.00E�00 0.00E�00

a Body weight at the time of challenge.b Calculated based on the estimated concentration of B. melitensis 16 M in the exposurechamber and the total volume of air inhaled by the RMs.




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levels in unchallenged animals (group 10) remained below thelevel of detection of 0.5 mg/dl throughout the 8-week period.These results were confirmed by CRP ELISA.

All groups challenged with B. melitensis had increased levelscompared to baseline of ALT for at least one time point postchal-lenge. While the increase in ALT was still within the normal clin-ical range, the trend differed from group 10 (unchallenged), sincethe control animals did not show any increase in ALT from base-line at any time point. The ALT levels in the challenged animalsremained elevated until day 56 postchallenge.

While remaining within the normal clinical range, the ALPlevels for the group 9 challenged animals trended toward in-creased levels by day 35 postchallenge, and levels were above theclinically normal range by day 42 postchallenge for one of thegroup 9 challenged animals. The ALP levels for unchallenged an-imals remained steady throughout the 8-week period.

LDH levels started to increase by day 21 postchallenge for oneof the animals challenged with B. melitensis 16 M, and by day 28both group 9 challenged animals had LDH levels above the clini-cally normal range. While the LDH levels remained elevated com-pared to the unchallenged animals, the levels for the group 9 chal-lenged animals did start to decline by day 35 postchallenge.

Clinical hematology. Overall, there were minimal changes inthe hematology parameters, aside from minor signs of anemia,which is similar to what is observed in human brucellosis patients(3). While there was a small decline in hematocrit and hemoglobinlevels in both challenged and unchallenged animals, the overalldecrease for challenged animals was more apparent (data notshown). While neutrophil levels were above the clinically normalrange for animals challenged with B. melitensis 16 M, levels werealso above this range for the unchallenged animals. White bloodcell counts were slightly above the clinically normal range fromdays 35 to 56 postchallenge for animals challenged with B. meliten-sis, but one of the unchallenged animals also had a white blood cellcount above the range on day 35.

Immune responses to brucellosis. A series of serological as-says, which are used clinically for diagnosis of human brucellosis(1, 4–6,19), were evaluated for each animal in phase II of thisstudy. These immunoassays included microagglutination, anti-Brucella IgG, and anti-Brucella IgM ELISAs. Inflammatory bio-markers were evaluated as well, which included neopterin andCRP ELISAs. Figure 3 illustrates the kinetics of the immune re-sponses to brucellosis of the two RMs not euthanized until the endof the study, compared with the two unchallenged controls. (The

FIG 1 Baseline-adjusted telemetry results for challenged and unchallenged animals. Telemetry data for expiratory time (A), activity (B), body temperature (C),respiratory rate (D), heart rate (E), inspiratory time (F), pulse pressure (G), systolic pressure (H), and diastolic pressure (I) are shown. Closed circles, averagesof two animals challenged with the targeted dose of 250,000 CFU B. melitensis; open circles, averages of two unchallenged animals.

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CRP ELISA showed the same trend as the results from the Advia ofelevated CRP values by day 21 postchallenge). In addition, theresults in Table 2 show that when all challenged animals wereincluded in the analysis, at least 50% of animals were positive forBrucella IgG, IgM, neopterin, and CRP titers by day 21 postchal-lenge.

A microagglutination titer was detected in the serum of all RMsby day 21 postchallenge. Positive microagglutination and anti-Brucella IgM titers were detected as early as day 14 postchallenge,while there was an animal with anti-Brucella IgG titers by day 7postchallenge. This animal was also positive for IgG titers prior tochallenge. While all challenged animals were positive by day 21

FIG 2 Clinical pathology kinetics for animals challenged with B. melitensis 16 M and for unchallenged animals. (A) AST; (B) CRP; (C) ALT; (D) ALP; (E) LDH.Closed circles, challenged animals; open triangles, unchallenged animals.




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postchallenge for microagglutination and anti-Brucella IgM, only50% of animals had positive IgG titers. These results suggest thatthe microagglutination assay and anti-Brucella IgM ELISA aremore sensitive indicators of seroconversion than the anti-BrucellaIgG ELISA.

Weekly ultrasound-guided liver biopsy and ultrasound im-aging of splenomegaly. Ultrasound imaging demonstrated thatbrucellosis caused splenomegaly in RMs (Fig. 4). In this study,

spleen size peaked at day 28 postchallenge, followed by a declineand then an increase in splenomegaly by day 42 postchallenge.

There were no positive bacterial cultures from liver biopsysamples until day 14 postchallenge. In phase I, 33% (2/6) of ani-mals on day 21 (1 group 2 and 1 group 3 animal), day 28 (1 group2 and 1 group 3 animal), and day 35 (1 group 1 and 1 group 2animal) had positive bacterial cultures from the liver biopsy spec-imens. By day 42 postchallenge, only one animal had a positive

FIG 3 Kinetics of immune responses for animals challenged with B. melitensis 16 M and for unchallenged animals. (A) Microagglutination; (B) anti-BrucellaIgM; (C) neopterin; (D) anti-Brucella IgG. Closed circles, challenged animals; open triangles, unchallenged animals.

TABLE 2 Number of phase II challenged animals positive for each immunoassay at each time point

Assay (criterion for positive result)

No. of animals positive/no. tested for assay on indicated day postchallenge

�5 7 14 21 28 35 42 56

Neopterin ELISA (titer � 6.25)a 0/10 0/10 2/10 4/8 5/6 3/4 3/4 0/2CRP ELISA (titer � 1.89)a 0/10 4/10 4/10 6/8 6/6 4/4 4/4 2/2Microagglutination (titer � 40)b 0/10 0/10 2/10 8/8 6/6 4/4 4/4 2/2Anti-Brucella IgG ELISA (titer � 12)c 1/10 1/10 2/10 4/8 5/6 3/4 4/4 2/2Anti-Brucella IgM ELISA (titer � 12)c 0/10 0/10 3/10 8/8 6/6 4/4 4/4 2/2a Positive based on means of all samples � 2 standard deviations.b Positive based on human clinical data.c Positive based on manufacturer recommendations.

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bacterial culture from the liver biopsy specimen. During phase II,two animals (25%; 2/8) were positive on day 14 postchallenge,while 67% (4 of 6) of animals were positive on day 21 postchal-lenge. On day 28, 50% (2 of 4) of the challenged animals remain-ing had a positive liver biopsy, and on day 35 postchallenge 75% (3of 4) of animals had positive liver biopsies. Both challenged ani-mals on day 42 postchallenge had negative liver biopsies (Table 3).

Terminal tissue burden. On day 56 postchallenge for thephase I animals, the highest bacterial burden was typically foundin the spleen (with all but one sample above 1.00E�02 CFU/g),

regardless of the challenge dose (Fig. 5A). Samples from the tissuesshown below the dotted line (mostly lung, liver, and kidney) wereconsidered positive, since the numbers of colonies fell below theLOQ (100 CFU/g) but above the LOD (3 CFU/g).

In phase II of this study, the tissue burden in the lung, liver,kidney, and spleen were highest on days 14 through day 28 post-challenge, with all samples above the LOQ of 1.00E�02 CFU/g(spleen bacterial load between 1.00E�04 and 1.00E�06 CFU/g),and then the bacterial burden decreased over time. Although pos-itive tissue burdens were detected in the day 56 postchallenge sam-

FIG 4 Kinetics of splenomegaly in animals challenged with B. melitensis 16 M. Representative ultrasound images of the spleen from an animal challenged withB. melitensis on day 14 (A), day 21 (B), day 28 (C), day 35 (D), or day 42 (E) postchallenge are shown. (F) The length of the spleen. Each line represents the spleensize for an animal that was challenged with B. melitensis 16 M.




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ples, the concentration was below the LOQ (Fig. 5B). The clearance ofthe bacteria from the tissues is consistent with a protective immuneresponse by the host. Immune clearance of the bacteria is supportedby the increases in microagglutination, anti-Brucella IgG and IgMtiters, and the reduction in inflammatory markers CRP and neop-terin by day 56 postchallenge. However, it is not known if the RMswould have developed a sterilizing immunity or if they would becomechronically infected with low levels of the bacteria.

Necropsy and histopathology. For phase I and phase II of thisstudy, no gross lesions were observed in any of the RMs. Table 4shows the incidence of Brucella-related microscopic observations,with average severity scores for animals that were euthanized onday 56 postchallenge. Animals euthanized prior to that time pointhad similar microscopic observations. Figure 6 shows representa-tive Brucella-related lesions in the spleen, liver, and kidney in aninfected animal.

Microscopic findings attributed to aerosol exposure to B.melitensis 16 M were present in the liver, lung, bronchial lymphnodes, kidney, and spleen of challenged animals in phase I andphase II. B. melitensis 16 M-induced lesions included minimal tomild hepatocellular necrosis, which occurred in the livers of allphase I challenged animals (6/6) and animals euthanized on days28 and 42 postchallenge in phase II. Necrosis in the liver was oftenassociated with sinusoidal leukocytosis and/or chronic/chronic-active inflammation (lymphocytes, macrophages [histiocytes],and neutrophils), which was present randomly (when associatedwith necrosis) or in periportal areas. Chronic inflammation wascharacterized by lymphocytes, plasma cells, macrophages, andfewer or no neutrophils, which generally surrounded portal areasof the liver. Chronic/chronic-active inflammation was also pres-ent in the kidney of 67% (4/6) of the phase I challenged animalsand 30% (3/10; 2/2 animals euthanized on day 14 and 1/2 animalseuthanized on day 28) of the phase II challenged animals. Eighty-three percent (5/6) of challenged animals in phase I and 80%(8/10) of challenged animals in phase II also had increased numbers

FIG 5 Tissue burden in animals challenged with B. melitensis 16 M. Phase I(A) and phase II (B) tissue burden was measured from spleen, liver, kidney,and lung tissue of each animal on the day of scheduled euthanasia. Closedcircles, spleen; open circles, liver; closed triangle, kidney; open triangle, lung.The dashed line represents the LOQ (100 CFU/g).

TABLE 3 Liver biopsy results

GroupTargeted challengedose (CFU)

Actual challengedose (CFU)

Biopsy result ona:

Day 7 Day 14 Day 21 Day 28 Day 35 Day 42

1 5.00E�03 5.44E�03 0 0 0 0 � �1 5.00E�03 6.10E�03 0 0 0 0 0 02 5.00E�04 3.18E�04 0 0 � � � 02 5.00E�04 4.02E�04 0 0 0 0 0 03 5.00E�05 4.68E�05 0 0 0 0 0 03 5.00E�05 5.11E�05 0 0 � � 0 04 0.00E�00 0.00E�00 0 0 0 0 0 04 0.00E�00 0.00E�00 0 0 0 0 0 05 2.50E�05 3.24E�05 0 NA NA NA NA NA5 2.50E�05 3.17E�05 0 NA NA NA NA NA6 2.50E�05 4.39E�05 0 � NA NA NA NA6 2.50E�05 3.07E�05 0 0 NA NA NA NA7 2.50E�05 4.30E�05 0 0 0 NA NA NA7 2.50E�05 3.48E�05 0 0 � NA NA NA8 2.50E�05 4.47E�05 0 0 0 � � NA8 2.50E�05 4.14E�05 0 0 � 0 0 NA9 2.50E�05 4.04E�05 0 0 � � � 09 2.50E�05 3.39E�05 0 � � 0 � 010 0.00E�00 0.00E�00 NA NA NA NA NA NA10 0.00E�00 0.00E�00 NA NA NA NA NA NAa NA, not applicable; �, positive for B. melitensis 16 M; 0, negative for B. melitensis 16 M.

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of neutrophils (polymorphonuclear cells) in the splenic sinusoidscompared to unchallenged animals, as well as minimal hyperplasia ofthe splenic white pulp (50% [3/6] in phase I and 90% [9/10] in phaseII). However, the splenic sinusoids of one unchallenged macaque alsocontained several neutrophils, so their relevance to B. melitensis 16 Mis uncertain. Chronic or chronic-active inflammation (diagnosed inareas that contained lymphocytes and macrophages but few or noneutrophils) was observed perivascularly in the lungs. The macro-phages in the lungs of phase II animals were often epithelioid in ap-pearance and contained finely granular eosinophilic cytoplasm occa-sionally forming microgranulomas. Bronchial and/or mediastinallymph nodes in 50% (3/6) of the phase I challenged animals and100% (10/10) of the phase II challenged animals had minimal to mildsinus histiocytosis.

Bone marrow was evaluated microscopically in sections ofsternum of Phase II animals. Erythroid cellularity was slightly in-creased in all exposed male macaques compared to the unchal-lenged male. However, bone marrow cellularity in exposed fe-males was similar to the unchallenged female, except that oneanimal had increased erythroid cellularity (or decreased marrowfat) and was diagnosed with mild erythroid hyperplasia. It is un-clear whether these bone marrow differences were related to B.melitensis 16 M, due to normal variation, or due to other relatedcauses (e.g., anemia). There was no evidence of inflammation(e.g., histiocytic/granulomatous or neutrophilic cellular accumu-lations) in the bone marrow of any animals. In addition, the kneejoint of challenged animals in Phase II was similar to the unchal-lenged animals.

Microscopic lesions were similar to previously described B.melitensis 16 M lesions in aerosol-exposed RMs (18), except that

neutrophils (polymorphonuclear cells) were more prevalent andnecrosis was associated with hepatitis in the livers of the animals inthe current study. Additionally, the testes were not affected in themacaques of the current study. Lesions occurred sporadically inthese macaques, and there was no apparent dose response betweenchallenged groups for phase I animals. For phase II animals, le-sions occurred sporadically in all macaques until day 42, whenmicrogranulomas were observed in the lung and liver of both themale and female macaques. However, microgranulomas were notpresent in the examined liver and lung sections at day 56, andchronic-active inflammation was decreased or absent in these or-gans at day 56. None of the lesions described above was present toany meaningful extent in unchallenged animals.

DISCUSSION

In order to develop an animal model of brucellosis that meets twokey requirements of the FDA “Animal Rule,” this study character-ized the pathophysiology of aerosolized brucellosis in RMs andlinked human immune diagnostic markers of infection to thisanimal model. The optimal challenge dose to be used to examinethe pathophysiology of inhalational brucellosis during an 8-weektime period was determined by examining the clinical parameters(telemetry) and bacterial load (weekly liver biopsies and tissueburden on the last day of in-life) during phase I of this study(targeted challenge doses ranging from 5.00E�03 to 5.00E�05CFU/animal). In addition to the nonspecific indicators of brucel-losis (i.e., clinical observations, body temperature, weights), clin-ical parameters directly associated with brucellosis were assessedin challenged animals. These assays were used to detect bacteria inblood cultures and bacteria in selected tissues (bacterial burdensin liver, lung, kidney, and spleen; liver biopsy specimens). Whilenone of the animals had a positive blood culture in phase I, bac-teria were present in the tissues of all animals (lung, liver, kidney,and/or spleen) after aerosol exposure to B. melitensis (day 56 post-challenge). In addition, all animals challenged with B. melitensiswere febrile and had splenomegaly during the postchallenge pe-riod. Therefore, all challenged animals in phase I were infectedwith B. melitensis.

While all animals challenged with the targeted doses of 5,000CFU, 50,000 CFU, or 500,000 CFU were infected with B. meliten-sis, a dose between 50,000 CFU and 500,000 CFU was determinedto be optimal for assessing infection over an 8-week time period(chronic infection). In addition, a time course study with a highchallenge dose of 250,000 CFU would be more indicative of whatmay occur during a bioterrorism incident. Therefore, 10 RMswere challenged with an average dose of 3.76E�05 CFU B.melitensis, and 2 animals remained unchallenged during the sec-ond phase of this study.

The changes observed in clinical pathology and hematologyparameters in this study were consistent with human brucellosis,in which the majority of clinical pathology indicators are indis-tinct (24). In addition, a recent study observed limited changes inclinical pathology parameters in RMs that were monitored for 7days after challenge (acute infection) with a high dose of5.60E�08 CFU B. suis (24). The only clinical pathology parame-ters that were affected in the current chronic infection study wereliver enzymes (LDH, ALT, AST, and ALP), which is consistentwith the mild increase in liver enzymes (LDH and ALP) that hasbeen observed in human brucellosis patients (3). An adverse effectof brucellosis on the liver in the current study was supported by

TABLE 4 Incidence summary of Brucella-related microscopicobservations with average severity scores

Tissue and observation

Total no.of animalsexamineda

Incidenceof thechange

Avgseverityb

LiverInflammation 8 8 1.6Sinusoidal leukocytes 8 5 0.9Necrosis 8 6 1.3

LungInflammation 8 5 0.8

Lymph nodeBronchial sinus histiocytosis 8 5 1.0Mediastinal sinus histiocytosis 8 1 0.1

SpleenInfiltration of neutrophils 8 7 1.4Hyperplasia (white pulp) 8 4 0.5

KidneyInflammation 8 5 0.6

Bone marrowHyperplasia 2 1 1.0

a Only animals that were euthanized on day 56 postchallenge are included in thissummary.b Lesions were graded on a scale of 1 to 4, as follows: 1, minimal, least detectable lesion;2, mild, easily discernible lesion; 3, moderate, change affecting large area of representedtissue with potential to be relevant; 4, marked, lesion that approached maximal.




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positive liver biopsies, liver tissue burdens, and microscopic ob-servations of hepatocellular necrosis.

In addition to a limited change in clinical pathology parame-ters, a limited effect on clinical hematology parameters was ob-served in the B. suis aerosol-challenged acutely infected RMs (24).This is consistent with the results from the current study. There-fore, clinical pathology and hematology trends are similar for bothacute (B. suis) and chronic (B. melitensis) brucellosis infection.There was a slight decline in hematocrit and hemoglobin levels inchallenged animals in the current study, which is consistent withthe mild anemia that has been observed in human brucellosispatients (3).

Similar to human serology biomarkers for diagnosis, animalsin the current study exhibited microagglutination and increased

anti-Brucella IgG, anti-Brucella IgM, neopterin, and CRP titerspostchallenge (1, 4, 5, 19). These biomarkers, which are regularlyused in the clinic to confirm human brucellosis, can be used infuture NHP studies as diagnostic triggers for treatment and toevaluate the efficacy of a potential vaccine or therapeutic agent ina treatment study. While it is unknown why one animal had anIgG titer prior to challenge, it could have been due to cross-reactivity specific to that animal or an unknown previous expo-sure to Brucella. However, this animal was negative for all othermarkers of infection prior to challenge (i.e., microagglutination,anti-Brucella IgM), and this animal developed a microagglutina-tion and anti-Brucella IgM titer at a time point similar to otherchallenged animals. Therefore, if this animal had a true IgG titerprior to challenge, it did not appear to alter the response to chal-

FIG 6 Brucella-related lesions in the kidney, liver, and spleen. (A) Magnification (40�) of Brucella-infected liver, showing hepatocellular necrosis. Normalhepatic architecture (plates, on the right) is disrupted by an area of coagulative necrosis (left) characterized by infiltrating inflammatory cells and hypereosino-philic cellular casts (arrows). (B) Magnification (40�) of unchallenged control liver with normal hepatic plates (arrows) and sinusoids (arrowhead). (C)Magnification (20�) Brucella-infected kidney with sclerosing (chronic active) inflammation (arrows) and dilated protein-filled tubules (arrowheads). Note:normal tubules are evident on the right above a normal glomerulus (star). (D) Magnification (20�) of unchallenged control kidney with normal tubules (arrows)and a glomerulus (star). (E) Magnification (40�) of Brucella-infected spleen. Nodular infiltrate of white blood cells composed of lymphocytes, and epithelioidmacrophages (arrowheads) and aggregates of polymorphonuclear cells (neutrophils) invading the red pulp are shown. A normal lymphoid follicle (star) is alsoevident. (F) Magnification (40�) of an unchallenged control spleen with a normal amount of white cell elements (predominantly mononuclear cells orlymphocytes [arrowheads]) in the red pulp (arrow). Hematoxylin and eosin stain was used for all tissues.

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lenge, as the animal had the same response as those without abaseline titer.

CRP, a major inflammatory marker, was significantly in-creased for animals that were challenged and euthanized after day21 postchallenge compared to the unchallenged animals. ThisCRP result is consistent with human brucellosis, in which CRPlevels are increased to high levels during brucellosis infection (5).In addition to changes in liver enzymes and CRP assessment,Brucella-specific indicators of illness (bacteremia, bacterial bur-dens, and splenomegaly) appear to be better indicators of brucel-losis infection, as opposed to nonspecific indicators (i.e., clinicalobservations and hematology), and should be used to supportdiagnosis of brucellosis in humans as well as NHPs.

A clinical profile for each animal was developed in order todefine the pathophysiological mechanism of toxicity associatedwith brucellosis over an 8-week time period. While challengedanimals had limited changes in hematology parameters, animalsdid have changes in certain clinical chemistry parameters, CRP,and body temperature typically between 3 and 5 weeks postchal-lenge. In addition, specific signs of Brucella illness in challengedanimals included positive blood cultures, positive liver biopsies,splenomegaly, and tissue burden for B. melitensis during the sametime frame. These results suggest that the 3-week postchallengetime period would be an appropriate time period to therapeuti-cally treat RMs in a future study.

Similar to human cases, animals appeared to mostly resolvebrucellosis infection by day 56 postchallenge, as evidenced by anegative blood culture and resolution of a fever and abnormalclinical chemistry and CRP levels. These animals still had somebacteria located in tissues, although these levels were below the

LOQ, suggesting that the bacteria were being cleared by the host’sadaptive immune response. However, the low levels of bacteriathat remained in some animals could result in relapse. The clinicalprofile observed in the challenged animals was not observed in theunchallenged animals (Fig. 7). This model mimics the chronicinfection observed in humans. Therefore, the RM brucellosismodel could be used to predict the response to therapeutics andvaccines in humans.

Similar to humans, all NHPs assessed in both phase I and phaseII, except for one animal, did not exhibit any abnormal clinicalsigns, except for minimal nonspecific observations (an occasionalstool abnormality or decreased appetite). Notably, from days 27 to30 postchallenge, one animal (female in group 9) was observed tobe weak. In addition, this animal lost weight, had changes in he-matological parameters, increases in liver enzymes (AST, ALT,and LDH), a positive liver biopsy, and increased CRP levels. Thisanimal was febrile during this time period as well. After day 30postchallenge, however, this animal was observed to be normal,and the hematological parameters and liver enzymes started todecline to levels closer to the baseline levels. This animal may havebeen more susceptible to brucellosis, which led to the abnormalclinical pathology and resulted in the more apparent abnormalclinical signs and weight loss. Interestingly, this animal appearedto recover from the infection, as there were only a few bacteriadetected in spleen tissue (below the LOQ) and all qualitative en-richment tissue was negative on day 56 postchallenge. In addition,the fever was resolved and the blood culture was negative by day 56postchallenge.

In addition to the nonspecific indicators of brucellosis, clinicalparameters directly associated with brucellosis were assessed in

FIG 7 Clinical profiles for an animal challenged with B. melitensis 16 M (A and C) and an unchallenged animal (B and D). (A and B) Solid lines with closed cricles,white blood cell count; dashed lines with closed diamonds, LDH levels; dotted lines with stars, CRP levels; solid lines, body temperature; triangles, number ofpositive liver biopsy specimens. (C and D) Solid lines with closed circles, microagglutination; dashed lines with closed diamonds, anti-Brucella IgG; dotted lineswith stars, anti-Brucella IgM; solid lines with triangles, neopterin.




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challenged animals. These assays were to detect bacteria in bloodcultures and in selected tissues (bacterial burden, liver biopsies).While all phase I animals had negative blood cultures for Brucella,50% (5/10) of phase II animals were bacteremic at the time ofeuthanasia. The difficulty of detecting bacteria in the blood inNHPs is parallel to what is observed in human cases. The percent-age of human brucellosis cases with positive blood cultures canrange from 15 to 70%, depending on the population and cultureconditions (21). The positive results observed in phase II wereprobably due to the enrichment of Brucella in the blood by sub-culture in broth followed by plating a sample of the enrichmentbroth on agar. While it has been shown that bacitracin and nali-dixic acid at the concentrations used in this study can reduce the B.melitensis CFU count, these supplements did prevent the growthof contaminants, and therefore, allowed for assessment of a posi-tive culture. Since the blood culture was only a qualitative obser-vation, the potential decreased number of CFU did not adverselyaffect the results (17). This enrichment method will be used infuture studies with blood samples in order to enhance the chancesof observing a positive culture. Unlike the blood samples, tissuessamples did not need to be enriched prior to plating on MTMagar. B. melitensis was identified in at least one tissue (lung, liver,kidney, and/or spleen) from each animal after aerosol exposure toB. melitensis at the time of euthanasia. Therefore, all challengedanimals were infected with B. melitensis. The largest B. melitensisburdens were found in the spleen, which is consistent with theresults reported by Mense et al. (18).

In addition to the clinical parameters, tissues from all animalswere examined both grossly and microscopically in order to definethe pathology of Brucella-infected NHPs over the 8-week timeperiod. Consistent with human disease, there were microscopiclesions associated with the liver, lung, spleen, lymph nodes, andkidney (16). The inflammation and necrosis observed in the cur-rent study were consistent with the findings reported by Mense etal. (18), who also studied B. melitensis aerosol-infected RMs, andwhat is observed in human brucellosis pathology.

Conclusions. Sixteen NHPs (phase I and phase II) were chal-lenged with B. melitensis 16 M by the inhalation exposure routeand monitored for clinical and physiological changes followingchallenge. Four NHPs remained unchallenged.

This study provides data supporting the use of RMs to test theefficacy of therapeutics to treat brucellosis, as this study defined signsof illness (body temperature, liver enzyme changes, CRP) that can beused independently or in conjunction with specific diagnostics (tissueburden, liver biopsies, enriched blood culture, and splenomegaly) tomonitor onset of disease. The pathophysiological kinetics of brucel-losis in RMs was well characterized in this study.

Thus, the results from this study support the use of the RM asan animal model for inhalational brucellosis, and the model issuitable for testing the efficacy of a novel vaccine or therapeuticagainst B. melitensis under the “Animal Rule.”

ACKNOWLEDGMENTS

This work was supported by the Transformational Medical Technologiesprogram through CBRNIAC contract number SP0700-00-D-3180, TaskCB-09-0018 Task 769 from the Department of Defense Chemical andBiological Defense program through the Defense Threat ReductionAgency (DTRA).

We thank Phyllis Herr-Calomeni, Amanda Molder, Jennifer Cross,Vanessa Little, Jonathan Luu, Neil Gibson, and Ryan Hamilton for their

outstanding technical assistance. We also thank Robert Krile for statisticalinput and Oscar A. Bermeo Blanco for implanting the telemetry units.

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