INTRODUCTIONBrucellosis is caused by Brucella species, which infect a wide rangeof mammalian hosts. The World Health Organization considersbrucellosis as one of the seven neglected zoonoses that contributesto the perpetuation of poverty (Maudlin and Weber-Mosdorf, 2006).Efforts to control brucellosis have been challenging, with recentdisease re-emergence and new endemic foci posing significant risksto human health (Pappas et al., 2006). Management of individualswith brucellosis is extremely complex owing to its wide spectrumof clinical manifestations, resulting in high initial treatment failureand significant risk of relapse (Buzgan et al., 2010; Franco et al.,2007; Pappas et al., 2006). Osteoarticular complications fromBrucella infection are the most common clinical manifestation, butsymptoms also include debilitating neurological, heart and liverdamage (Buzgan et al., 2010). In a recent study, as many as 46.5%of brucellosis patients experienced osteoarticular complications(Turan et al., 2011). Often, the challenges of clinical brucellosis areassociated with its focal involvement; however, thepathophysiological manifestations in animal models of brucellosisremain poorly characterized.

Murine models have provided many cellular and molecularinsights into brucellosis pathogenesis, such as the efficacy of

vaccine candidates, nature of major virulence factors, and essentialhost immune responses (Baldwin and Goenka, 2006; Murphy etal., 2001). Pathology has been largely determined by the recoveryof Brucella colony-forming units (CFU) in well-studied organs suchas liver and spleen (Baldwin and Parent, 2002; Enright et al., 1990;Izadjoo et al., 2008; Mense et al., 2001; Silva et al., 2011; Young etal., 1979). Because of the wide tissue tropism observed in naturalinfections (Madkour, 1989), we hypothesized that murinebrucellosis also has a multifocal presentation.

We chose to identify and characterize novel infectious foci inBrucella-infected BALB/c mice using in vivo bioluminescenceimaging (BLI) in combination with guided histologicalcharacterization of the infected sites. BLI allows spatiotemporalidentification of infectious foci in living hosts (Contag et al., 1995;Doyle et al., 2004). This technique allows us to decisively indicatethe presence of metabolically active bacteria in deep tissues of mice(Hardy et al., 2009).

RESULTSBrucella melitensis multifocal dispersion in the skeletal structureof the murine hostBrucella has a complex interaction with the host, and multipleorgans and tissues can be affected in brucellosis patients, andfrequently result in osteoarticular complications. Infection of micehas been widely used as a quantitative model of Brucella infection;however, the extent of peripheral murine tissue colonization is notwell characterized. We evaluated bacterial dispersion in mice usinga virulent, bioluminescent strain of Brucella melitensis, which wepreviously characterized (Rajashekara et al., 2005). BLI signals weremonitored after intraperitoneal infection, and we observed rapidBrucella dissemination throughout the body (Fig. 1). Within days,BLI signals from the mouse body, mainly from peritoneum andliver (Fig. 1A), disseminated to peripheral sites, including the tail(Fig.  1B,C). Surprisingly, temporal resolution of infection in theperipheral sites was disproportionate and revealed a dispersion

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Disease Models & Mechanisms 6, 811-818 (2013) doi:10.1242/dmm.011056

1Department of Pathobiological Sciences, School of Veterinary Medicine,University of Wisconsin-Madison, Madison, WI 53706, USA2Albert Einstein College of Medicine, Departments of Medicine (Rheumatology),and Microbiology and Immunology, Bronx, NY 10461, USA*,‡These authors contributed equally to this work§Authors for correspondence (Vyacheslav.Adarichev@einstein.yu.edu;Splitter@svm.vetmed.wisc.edu)

Received 3 October 2012; Accepted 7 March 2013

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Brucellosis, a frequent bacterial zoonosis, can produce debilitating chronic disease with involvement of multiple organs in human patients. Whereasacute brucellosis is well studied using the murine animal model, long-term complications of host-pathogen interaction remain largely elusive. Humanbrucellosis frequently results in persistent, chronic osteoarticular system involvement, with complications such as arthritis, spondylitis and sacroiliitis.Here, we focused on identifying infectious sites in the mouse that parallel Brucella melitensis foci observed in patients. In vivo imaging showed rapidbacterial dispersal to multiple sites of the murine axial skeleton. In agreement with these findings, immunohistochemistry revealed the presenceof bacteria in bones and limbs, and in the lower spine vertebrae of the axial skeleton where they were preferentially located in the bone marrow.Surprisingly, some animals developed arthritis in paws and spine after infection, but without obvious bacteria in these sites. The identification ofBrucella in the bones of mice corroborates the findings in humans that these osteoarticular sites are important niches for the persistence of Brucella

in the host, but the mechanisms that mediate pathological manifestations in these sites remain unclear. Future studies addressing the immuneresponses within osteoarticular tissue foci could elucidate important tissue injury mediators and Brucella survival strategies.

Osteoarticular tissue infection and development ofskeletal pathology in murine brucellosisDiogo M. Magnani1,*, Elizabeth T. Lyons2,*, Toni S. Forde2, Mohammed T. Shekhani2, Vyacheslav A. Adarichev2,‡,§ and Gary A. Splitter1,‡,§

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pattern that is consistent with the segmented skeletal structure ofthe tail, with residual bacteria surviving for at least 2 weeks post-infection (Fig.  1A, day 6, day 13, arrow). Interestingly, thesepersistent foci were associated with defects in axial skeleton thatwere detected as dark spots in the tail (Fig. 1A, day 3). We furtherestimated the number of viable bacteria in caudal tissue segments(days 5 and 13 post-infection), and the BLI intensity from the caudalregion strongly correlated with the bacterial CFU counts (Fig. 1D;R2=0.84). The CFU counts were directly proportional to the BLI,confirming that the observed luminescent pattern correspondedto the level of viable and replication-competent bacteria in thetissues. Although most infected animals did not display noticeabledisease symptoms in the peripheral joints, some animals presentedredness and swelling, with visible lesions accompanying thepresence of bacteria, as shown by BLI (supplementary material FigsS1, S2, arrows).

Monitoring infection by BLI revealed initially the rapid bacterialdispersion dynamics throughout the mouse body followed by apunctuated luminescence pattern in the tail that matched theanatomical skeletal organization of vertebrae, suggesting directcolonization of osteoarticular tissue (Fig.  1B). Spatiotemporalevaluation of infectious foci revealed BLI signal in tails for at least

2 weeks after infection (Fig.  1). Interestingly, the BLI signalspersisted at a time (day 14) that bacteria were rapidly being clearedfrom the region corresponding to spleen and liver (Fig. 1A,C). Atthis time point (2 weeks after challenge), mice infected under similarconditions had ~3×105 CFU/spleen. Osteoarticular infections wereidentifiable and consistent in mice infected with a range ofinfectious inoculum doses (Fig. 1; supplementary material Fig. S1).

To investigate the mechanisms of bacteria dissemination, wesought first to characterize the relevant osteoarticular sites thatparallel those affected in the disease in humans, possibly colonizedby Brucella. Imaging at the higher BLI spatial resolution (4 binning,10 cm field-of-view; see Materials and Methods) showed thatBrucella-positive skeletal structures also include bones of the kneejoints as well as smaller bones of the paws (Fig. 2). BLI patternswere remarkably symmetrical for the right and left knees, pawsand ventral processes of the axial caudal skeleton, thereforehighlighting the non-random pattern of bacteria disseminationroutes in the body. Additionally, and as expected, the earliest andstrongest BLI signal observed was from the liver (Fig. 2, ‘L’). Usinghigh-resolution imaging, we identified five major locations ofBrucella-associated cells in the mouse skeleton: (1) tibia and femurbones of the knee joint; (2) calcaneus; (3) metatarsophalangeal andmetatarsotarsal articulations in paws; (4) ankle aspect of the tibia;(5) ventral processes of caudal vertebrae specifically inprezygapophyses, but not in transverse processes.

Histological localization of Brucella in liver, spleen and skeletaltissuesBrucellosis patients frequently display symptoms associated withbone and joint infection (Buzgan et al., 2010). After identifyingpotential skeletal infectious foci in mice using BLI, we sought todefine the specific histological location of bacteria. To characterizethe rheumatologic involvement of mice following Brucella infection,histopathological analysis was performed on small joints of hindand fore paws and larger knee and hip joints. Axial skeleton fromthe thoracic spine to caudal region, including the entire sacrum,was also examined.

To monitor Brucella dissemination at the tissue level,immunohistochemical (IHC) staining with antibodies to Brucella(Cat. # TC-7011, Tetracore Inc.) was used. Cross-reactivity ofsecondary antibodies with endogenous immunoglobulins couldproduce a considerable background, particularly when IHC ofspleen and bone marrow tissues was performed. Specifically forthis reason, we selected an alternative IHC detection approach forBrucella: primary antibodies were biotinylated and detected withstreptavidin conjugated to horseradish peroxidase (St~HRP). Whenprimary and secondary antibodies are used, it is most effective toblock tissues by using normal serum from the same host speciesas the labeled secondary antibody. Because secondary antibodieswere not used for the detection in our protocol, the major concernwas blocking endogenous biotin in spleen, bone and marrow tissuesto prevent nonspecific staining with St~HRP conjugate. Several setsof control staining were used to prevent false-positive signals. First,the complete IHC protocol was performed with tissues of mice thatwere not infected with Brucella, and produced no backgroundstaining. Second, primary goat antibodies were substituted withnormal goat serum and then applied to St~HRP detection (Fig. 3).Non-specific control staining was negative in liver (Fig. 3), bone

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TRANSLATIONAL IMPACT

Clinical issueInfection by Brucella spp results in brucellosis, a common, highly contagiouszoonosis. Brucella can induce multiple effects in the host and, in humans,infection frequently results in persistent, chronic osteoarticular complications,including peripheral arthritis, spondylitis and sacroiliitis. In addition, thepathogen is able to persist in the bone marrow, resulting in chronic infectionof the host. Although acute brucellosis has been extensively studied usingmurine models, the focal complications remain poorly understood because theextent of the infection in the many potential host sites (e.g. peripheral joints)cannot be easily evaluated in living animals.

ResultsHere, the authors use a bioluminescent strain of Brucella melitensis to monitordispersion of the bacteria in osteoarticular tissues of living BALB/c mice,followed by histological characterization of the infection sites. In vivo imagingdemonstrated the rapid dispersion of bacteria to multiple sites in the skeletalstructure of the murine host, following Brucella infection. Furthermore, theauthors’ histological analysis showed that Brucella-positive cells wereconsistently located in the marrow regions of all bone types studied. Miceinfected for a chronic period of 26 weeks exhibited multiple skeletalcomplications with inflammatory and non-inflammatory features.

Implications and future directionsIndividuals with brucellosis display a wide spectrum of clinical manifestations.The high rate of treatment failure and relapse associated with the disease isoften attributed to the intracellular survival of bacteria at the foci of infection.The identification of B. melitensis in murine bone marrow corroborates thetheory that this site is an important niche for chronic persistence of bacteria inthe host. It remains unclear whether infection at this site is facilitated byimmune tolerance mechanisms, or, conversely, whether the immune responsescause inflammation and mediate the bone pathological manifestationsobserved. Therefore, future studies addressing the immune responses at theosteoarticular tissue foci will provide insight into the long-term survivalstrategies of Brucella and resulting chronic tissue injury in human patients. Thisstudy demonstrates the utility of the murine model for the exploration ofpathophysiological mechanisms underlying chronic brucellosis.

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marrow (Fig. 4) and spleen (not shown). Also, St~HRP stainingalone was used while omitting the primary antibodies, and stainingwas negative in all studied tissues (data not shown).

Liver infection with B. melitensis was associated with massivegranuloma formation with fusion of individual granulomas aspreviously observed (Ko et al., 2002a; Ko et al., 2002b) (Fig. 3). Thegranuloma core consisted of round mononuclear cells with enlargednuclei and cytoplasm. Core cells were surrounded by smallerepithelioid-like elongated macrophages (Fig. 3A,C). As expected,IHC-positive staining for Brucella confirmed the presence ofbacteria in the granuloma core (Fig. 3A,B,E). Higher magnification(1300×; Fig. 3E) indicated the presence of Brucella antigen in thecytoplasm of large round mononuclear cells and the absence ofbacteria in epithelioid macrophages (Fig.  3B vs 3C). Brucella-positive signal was located solely inside granulomas, further

supporting IHC specificity. Taken together, the livers of infectedmice had Brucella mainly in the granuloma core and many fewerbacteria in Kupffer residential macrophages. Brucella was alsodetected in the spleen red pulp of acutely infected mice, whereasthe white pulp was negative (not shown). Granuloma formationwas not observed in bone marrow or spleen.

After 3 weeks of infection, bone marrow of all studied bones –including small bones of the hind and fore paws, tibia, femur, ileum,sacrum, vertebral bodies and processes – were examined forBrucella (Fig.  4). Interestingly, Brucella-positive cells wereconsistently located in the marrow of epiphyseal and metaphysealregions and not detected in diaphyseal regions (Fig. 4B). Similarly,in vertebrae, homologous bone parts were infected that includedendplates and subchondral/sub-growth plate areas (Fig. 4A,E,F).Articular cartilage of synovial joints, synovial cavity, nucleus

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Fig. 1. Distribution of Brucella melitensis in BALB/c mice following i.p. infection. BALB/c mice were inoculated intraperitoneally with 1×107 CFU and imagedat the designated time points. Color scale represents bioluminescent signal intensity. (A)One of four animals is shown, on selected days. Bacteria disperse fromthe injection site (lower abdomen) to reach peak luminescence levels around day 6 and then are progressively cleared from the animal. A representative of thearea used for quantification of photonic intensity – region of interest (ROI) – is depicted in the figure. Arrow indicates residual bacterial. FOV, field of view.(B)Closer field of view (FOV; 10 cm) reveals punctuated diffusion of light in the lower limbs and tail of acutely infected mice (day 5; one of three animals isshown). Dispersion pattern is consistent with the segmented skeletal structures of these sites. (C)Spatiotemporal quantification of luminescence dispersal andpersistence in body (blue) and tail (red) regions (ROI) of four infected BALB/c mice. Each line represents BLI luminescence quantification from an individualmouse (photons/second/ROI/mouse). Note that the caudal luminescence pattern is more variable than the light dispersion in the animal body. (D)Distribution ofluminescence intensity (photons/second) and colony forming units (CFU) in 1.5-cm tail segments of seven BALB/c mice (24 segments total) at day 5 (threeanimals) and day 13 (four animals) post-infection. The solid line represents the linear regression of log CFU vs log photons/second/segment, and the dotted linesshow the 95% confidence interval of the fit.

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pulposus and annulus fibrosus of spinal discs were negative forBrucella (Fig.  4). Similarly, in bone marrow, ionized calciumbinding adaptor molecule 1 [IBA-1; a marker specific for membraneruffling and phagocytosis in macrophages (Ito et al., 2001)] and

Brucella IHC staining both exhibited a subchondral location(Fig.  4C), but only a small subset of IBA-1-positive monocytesincorporated Brucella (Fig.  4B vs 4C). Bone marrow of bothinfected and naïve mice contained large numbers of IBA-1-positivecells. Although additional blocking is not required (Invitrogen LifeTechnologies), a generic blocking buffer (5% normal donkey serum,2% BSA in 1 × PBS) was used to warrant rigorous specific bindingconditions for the primary antibodies.

Skeletal pathology in infected miceNaïve BALB/c mice rarely develop spontaneous inflammation insynovial joints (Adarichev et al., 2008; Farkas et al., 2009). BALB/cfemales infected with B. melitensis for 26 weeks exhibited multipleskeletal involvement with inflammatory and non-inflammatoryfeatures. Massive infiltration of inflammatory cells was found insynovial joints of the hind paws (Fig. 5A-C). Developing pannusand bone erosions were prominent features of the brucellosis-induced paw and spine arthritis, whereas Brucella itself was notdetected in any examined tissues by 26 weeks post-infection.Another site of inflammation was facet joints of the dorsal vertebralprocesses (Fig.  5D). In both paw and spinal arthritis, leukocyteinfiltration and bone erosions were marked. Also, torsionmisalignment of the axial skeleton was observed in the caudalregion, which was not accompanied with obvious inflammation(data not shown). This complex brucellosis-induced pathology withsterile synovitis resembles reactive arthritis andspondyloarthropathy in human patients.

DISCUSSIONIn this study, we show rapid systemic distribution of Brucellamelitensis in wild-type BALB/c mice, with high bacterial loads inmultiple infectious sites associated with liver, spleen, bone marrow,spine, bones and joints. Bioluminescent imaging revealed amultitude of infectious foci in the caudal axial skeleton, andinfected foci were corroborated using IHC staining specific for

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Fig. 2. High-resolution imaging of multi-focal dispersion of acute B.melitensis infection. With in vivo bioluminescent imaging at day 4 post-infection, Brucella was mainly localized intraperitoneally (IP), in the liver (L),knee (K), near ankle joint (A), metatarsophalangeal (MP), metatarsotarsalarticulations (MT), calcaneus (C) and ventral processes of caudal vertebrae(Ca). Note the mirrored patterns of Brucella distribution in the left and rightpaws and knees. Color scale represents bioluminescent signals intensity.Arrows indicate formerly uncharacterized sites of bacterial infection in knees,paws and tail. Image taken at 1-minute integration time and four binning forhigher-resolution setting.

Fig. 3. Brucella melitensis infectious foci in liver. At 3 weeks post-infection, pathogenic B. melitensis-infected cells developed multiple granulomas (A,D; H&Estaining). Immunohistochemical analysis of bacterial foci was performed using biotinylated primary antibodies (1.0 μg/ml, #TC-7011, Tetracore Inc.), endogenousbiotin blocking, and streptavidin-conjugated to horseradish peroxidase (St~HRP) using 3,3�-diaminobenzidine (DAB) as the final chromogen (B,E; brownprecipitate). Staining of normal liver of healthy non-infected mice produced no signal (not shown). Staining of liver tissue at 3 weeks post-infection using thesame protocol but substituting biotinylated anti-Brucella antibodies with normal serum produced no signal (F). Immunohistochemical analysis of macrophagelineage cells using antibodies to IBA-1 (dilution 1:500, #019-19741, Wako Chemicals USA, Inc.) followed by HRP rabbit polymer conjugate (#87-9263, InvitrogenLife Technologies) revealed positive staining of cells at the granuloma periphery (C). Residential macrophages – Kupffer cells were also positive for IBA-1 staining(C). Although Brucella was found in the granuloma core, these core cells were basically negative for IBA-1 staining (compare B versus C). Sections werecounterstained with hematoxylin (B,C,E,F). Scale bars: 20 μm (A,B,C,E), 50 μm (F) and 200 μm (D).

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Brucella. This extensive bacterial dissemination in the murine hostraises novel possibilities for use of this brucellosis experimentalmodel. Osteoarticular complications are particularly common inBrucella-infected humans, and the mouse model of brucellosis isparticularly useful for using guided imaging techniques to identifyinfectious osteoarticular foci.

Osteoarticular involvement in mice is progressive anddemonstrates a marked time lag after the peak of infection, thusresembling reactive arthritis in humans. The absence of bacteriais similar to observations of osteoarticular degeneration in some

humans previously infected by Brucella (Tuna et al., 2011). In astudy of 530 Brucella-infected patients, sacroiliitis was identifiedin 63 patients and 19 of these were culture negative (Ariza et al.,1993). In humans, Brucella-induced sacroiliitis is usually associatedwith acute spinal involvement (Ariza et al., 1993), whereasspondylitis, as we observed, is associated with a more chronic formof infection, with a median time of 9 weeks for spondylitis inhumans (Dayan et al., 2009).

Additionally, we demonstrate that, in acute infection, numerousgranulomas developed in livers by day 7. Morphologically, at leastthree cell types were readily identified in these granulomas: largecore cells positively immunostained for Brucella, epithelioidmacrophages, and, in the most outer layer of the granuloma,inflammatory lymphocytes and granulocytes. Over the course ofmonths after Brucella infection, granulomas lost the outerseemingly pro-inflammatory layer of cells. Also, some sporadicremnant granuloma cores possessed very weak positivity for IHCBrucella lipopolysaccharide (LPS) staining. Whether these cellsharbored live bacteria or only retained bacterial antigens isuncertain; however, live Brucella has been demonstrated to persistin BALB/c mice for greater than 360 days (Durward et al., 2012).

Recent efforts have initially characterized molecular aspects ofBrucella-induced arthritis and inflammatory bone loss using in vitrocell models (Delpino et al., 2012; Scian et al., 2011). Brucella cellinfection was found to influence bone metabolism via innateimmune sensing, in both human and mouse cells (Delpino et al.,2012), reinforcing the idea that studies of the murine Brucella-infected osteoarticular system will yield valuable insights into thehuman disease. The intra-articular injection of heat-killed Brucellafurther suggests that joint infection can induce a pro-inflammatoryenvironment (Scian et al., 2011). However, the nature of the cellularand inflammatory responses in vivo following infection remains tobe tested. Our findings suggest that macrophages as well as othercells of the bone marrow and osteoarticular sites are in contact

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Fig. 4. Histology of Brucella bone marrow foci in mice. Immunohistochemical detection of Brucella foci in bone marrow using modified avidin, biotinylatedenzyme complex (ABC) method (see legend for Fig. 3 and Material and Methods section). In skeletal tissues at 3 weeks post-infection, Brucella was located in thesubchondral space of vertebrae bone marrow (A,C,E,F) and bones (B). Despite the presence of bacteria, no skeletal pathology was found in mice at 3 weeks post-infection. Substitution of primary antibodies with normal goat serum and then ABC detection produced no signal (D). In bone marrow, IHC-Brucella staining wasrepresented by loose groups of positive cells in the subchondral area, the distribution of which was considerably different from the pan-marrow dispersion ofIBA-1-positive macrophage marker (C). Slides were counterstained with hematoxylin. AF, annulus fibrosa; GP, growth plate; NP, nucleus pulosus; T, trabecula.Scale bars: 200 μm (A-C) and 20 μm (D-F).

Fig. 5. Arthritis in post-infected mice. At 26 weeks post-infection, infectedBALB/c female mice developed arthritis in ankle joints of paws (A-C) and infacet joints of the spine (D). Massive inflammatory cell infiltration inducederosions of bones and vertebral processes (green arrows). Staining was withalcian blue and fast red (A,B,D) or H&E (C). Scale bars: 200 μm.

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with bacteria in vivo and, therefore, it would be important todetermine their role in the pathogenesis of osteoarticularbrucellosis.

Whereas brucellosis-induced arthritis in wild-type mice is aprogressive disease with postponed onset, mice carrying interferongamma deficiency (IFNγ KO) develop arthritis much earlier, whenlive Brucella is still present at high concentration in the body(Skyberg et al., 2012). Similar to our results, this type of acuteinflammation in IFNγ KO mice was limited to paw joints and tails.Interestingly, inflammation and Brucella foci were independent ofinfection route, suggesting that the osteoarticular site is a preferredlocation for bacterial persistence in the host and the mostinflammation-susceptible structure (Skyberg et al., 2012). Thesestudies are complementary, and suggest that murine fibroblast-likesynoviocytes and monocytes in joints can harbor bacteria and elicitinflammatory mediators following infection. It was shown recentlythat, at least upon direct infection in vitro with Brucella abortus,human fibroblast-like synoviocytes are able to maintain bacteriareplication and increase production of joint-damagingmetalloproteases (Scian et al., 2011).

Infection of mice with 107 CFU of bioluminescent bacteriaincreases the numbers of exposed permissible host sites for bacteriallocalization, increases peripheral sites having detectable levels ofbacteria by in vivo imaging, and provides reproducible infectiondevelopment in peripheral tissues. Infection of mice with lowernumbers of bacteria (5×103 and 1×104 CFU) also causesdevelopment of chronic localization of bacteria in tail vertebraljoints by day 28 (Rajashekara et al., 2005), supporting chronicinfection of this site with lower numbers of bacteria. Similarly, alow infectious dose (2×104) of B. abortus has been used in IFNγ−/−

mice to induce articular infection at day 42 (Skyberg et al., 2012).However, the frequency of articular infection in these immune-deficient mice was not determined.

The identification of Brucella in the murine osteoarticular tissuesubstantiates the notion that this site is an important niche forbacterial persistence and pathogenesis. In human osteoarticularinvolvement, spondylitis is typically observed in the lower lumbarregion, whereas arthritis is most prevalent in the knees (Madkour,1989). Similarly in our studies, the caudal vertebrae were the mostfrequently affected axial skeletal structures, and knees were alsocommonly infected. Future studies addressing the immuneresponses at the osteoarticular tissue foci might elucidate importanttissue injury mediators and Brucella survival strategies.

Bioluminescent imaging provides visual localization ofosteoarticular brucellosis and permits the further study ofinfiltrating host cells, cytokines and pathological changes occurringkinetically at these sites. BLI is sensitive to tissue oxygen levels,depth and density, perhaps limiting certain applications. Therefore,it remains possible that other sites harbor Brucella in the host. Wecould not detect Brucella BLI signal from femur heads, sacrum andcervical, thoracic or lumbar spine, but anticipate these sites aspotentially infectious foci in vivo. The survival, propagation anddissemination of bacteria to multiple organs results in severe clinicalmanifestations in brain, heart, liver and osteoarticular tissues.Understanding interactions of Brucella with the host at thesediverse infectious foci could elucidate important bacterial tissue-specific pathogenic mechanisms and niches that conceal bacteriaand contribute to brucellosis-induced complications. Our

consistent observation that Brucella can colonize these joints inthis experimental model might help to fill an important knowledgegap by providing the remarkable opportunity to explore the mousemodel in novel ways. For instance, focal progression of the infectionin peripheral tissues of human patients is not understood. Furtherexploration of this model should allow us to determine the relevantparallels to human clinical outcomes.

MATERIALS AND METHODSBacterial strains, growth conditions and preparation of inoculaBioluminescent B. melitensis (strain GR023) was previouslyconstructed by insertion of an EZ::TN transposon containing apromoterless lux operon in gene BMEI0101 of B. melitensis 16M(ATCC23456) (Rajashekara et al., 2005). This strain replicates invivo with kinetics and virulence similar to the parent wild-typestrain (Rajashekara et al., 2005). Brucella was maintained onbrucella agar (Becton Dickinson) and grown in brucella broth.Cultures started from isolated colonies were grown for 2 days inbrucella broth at 37°C and used for mice infection experiments.Bacterial concentration was estimated indirectly by measuring theoptical density at an absorbance of 600 nm and calibrating to knownstandard curve values. Prior to infection, samples were diluted inphosphate-buffered saline (PBS) for a final volume of 0.1-0.2 mlper mouse.

Mouse experimentsThis study was carried out in strict accordance with therecommendations in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health. The protocol wasapproved by the Institutional Animal Care and Use Committee ofthe University of Wisconsin-Madison, who evaluates the ethics ofanimal experiments (Permit Number: V00554). All in vivo imagingwas performed under isofluorane gas anesthesia. All efforts weremade to minimize animal suffering, and animals were housed inAssociation for Assessment and Accreditation of LaboratoryAnimal Care (AAALAC)-accredited facilities. Animals received astandard rodent diet and water ad libitum.

Groups of BALB/c females (n=36) (Harlan, Indianapolis, IN, andCharles River Laboratories) were infected intraperitoneally with 107

bacteria (or indicated dose in the text). This inoculum dose permitsdetectable levels of bacteria by in vivo imaging, and providesreproducible infection development in peripheral tissues. The dayof inoculation was designated day 0 and tissue was harvested at 3,6, 9, 12, 26 and 27 weeks post-infection. Bioluminescence imageswere obtained using an IVIS 100 system (Caliper Biosciences) withanimals under isofluorane gas anesthetic, administered at ~1-2%in oxygen. Images had an integration time of 1-10 minutes, binning1-16, field of view 10-20 cm. Higher-sensitivity images, meaninghigher ability to capture luminescence, were taken with longerintegration time and greater binning, whereas higher-resolutionimages, meaning more pixels per square inch, were taken withshorter integration time and lesser binning. Higher resolutionallowed pinpointing the source of luminescence, but capturing lesssignal. Data analyses were performed using Living Image softwareversion 3.2. Bioluminescence in a desired anatomical region wasdetermined by outlining the desired anatomical region followedthe Living Image software defining the photons per region ofinterest (ROI). Luminesce intensity is expressed as a calibrated

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measurement of the photon emission from the subject(photons/second/cm2/steradian). For CFU determination, seriallydiluted tissue lysate prepared from 1.5  cm tail segmentshomogenized in 0.1% Triton X-100 (in water) with a tissue grinder(VWR) was plated in brucella agar Petri dishes. The CFU counts/tailsegment was estimated as the number colonies recovered adjustedfor the corresponding dilution factor.

Histopathology and immunochemistryFor histopathological analysis, hind limbs from digits to femur,including knee joint, were collected. Also, axial skeleton fromthoracic to caudal regions was harvested. Tissues were fixed in 10%neutral buffered formalin, decalcified using Immunocal reagent(Decal Chemical Corp.) and embedded in paraffin at the AlbertEinstein College of Medicine Histopathology and ComparativePathology Facility, Bronx, NY. Tissues were sectioned at a thicknessof 5 μm and stained with hematoxylin and eosin (H&E) or alcianblue and nuclear fast red for histological confirmation of thearthritis.

For IHC analysis of bacteria, IgG antibodies to Brucella (#TC-7011, Tetracore Inc.) were labeled using N-hydroxysulfosuccinimideester of biotin (#21425, Thermo Fisher Scientific Inc.) accordingto the manufacturer’s protocol. After desalting, the degree of biotinincorporated into antibodies was measured using avidin conjugatedto 4�-hydroxyazobenzene-2-carboxylic acid (HABA; #21425,Thermo Fisher Scientific Inc.). Paraffin-embedded sections wereheated in a drying oven at 60°C for 55 minutes and deparaffinizedwith xylene and then rehydrated using 100-30% gradient ethanolbaths. Antigen retrieval for IHC Brucella cell-wall detection wasachieved with 0.25% trypsin (Mediatech) incubation for 25 minutesat room temperature. Slides were rinsed with 1×PBS andendogenous peroxidase activity was blocked by incubation in 0.3%hydrogen peroxide in 1×PBS for 5 minutes. Nonspecific bindingwas blocked for 1 hour at room temperature using 2% BSA (Sigma),2% normal non-immune donkey serum (Vector Labs), and 0.05%Tween-20 in 1×PBS. Sections were rinsed briefly with 1×PBS andadditionally blocked using streptavidin-biotin blocking kit (#SP-2002, Vector Labs). Finally, slides were incubated overnight at 4°Cwith biotinylated antibodies to Brucella (1.0  μg/ml; #TC-7011,Tetracore Inc.). After three washes for 5  minutes with 1×PBScontaining 0.05% Tween-20 (PBST), sections were incubated withstreptavidin conjugated to horseradish peroxidase (St~HRP; Pierce,Thermo Fisher Scientific Inc.) for 1 hour at room temperature. Afterthree washes with PBST, slides were finally stained with ImmPact3,3�-diaminobenzidine (DAB; Vector Labs) and counterstained withhematoxylin (Vector Labs) or alcian blue (Thermo Fisher ScientificInc.). Sections were dehydrated and mounted with Permount(Thermo Fisher Scientific Inc.).

For IHC detection of the IBA-1 marker, paraffin-embeddedsections were deparaffinized in xylene followed by rehydration ingraded alcohols similar to the protocol described for IHC detectionof Brucella. Antigen retrieval was performed for 20 minutes in 10mM sodium citrate buffer at pH 6.0 heated to 96°C. Endogenousperoxidase activity was quenched using 3% hydrogen peroxide in1×PBS for 10  minutes. Blocking was performed by incubatingsections in 5% normal donkey serum with 2% BSA in 1×PBS for 1hour at room temperature. The primary antibody to IBA-1 (#019-19741, Wako Chemicals USA, Inc.) was used at 1:500 for 1 hour

at room temperature. After rinsing with PBST, sections wereincubated with SuperPicture HRP rabbit polymer conjugate (#87-9263, Invitrogen Life Technologies) for 15  minutes, rinsed withPBST and developed using DAB as the final chromogen.Immunostained sections were lightly counterstained withhematoxylin mounted with Permount (Thermo Fisher ScientificInc.).

Statistical analysisData graphs were generated by using the statistical software Prism(GraphPad Software, Inc.), and statistical tests are indicated whenappropriate. Analysis of Pearson’s correlation was performed usingstatistical package SPSS v15.0 (SPSS, Inc.). P-values of <0.05 wereconsidered significant.ACKNOWLEDGEMENTSWe thank Tetracore for the provided antibody reagents and Dr Leandro Teixeira forthe critical review of this work.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSConceived and designed the experiments: D.M.M., V.A.A. Executed experimentsand acquired experimental data: D.M.M., E.T.L., T.S.F., M.T.S. Analyzed andinterpreted the data: D.M.M., E.T.L., M.T.S., T.S.F., V.A.A., G.A.S. Wrote the paper:D.M.M., V.A.A., G.A.S. Read, commented and approved the final version of themanuscript: D.M.M., E.T.L., T.S.F., M.T.S., V.A.A., G.A.S.

FUNDINGThis work was supported by National Institutes of Health (NIH) grants R01-AI073558, R01-AR053877, R21-AI088038 and BARD grant US-4378-11.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.011056/-/DC1

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