© 2014. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2014) 7, 1111-1122 doi:10.1242/dmm.015453

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ABSTRACTTuberculous meningitis (TBM) is one of the most severeextrapulmonary manifestations of tuberculosis, with a high morbidityand mortality. Characteristic pathological features of TBM are Richfoci, i.e. brain- and spinal-cord-specific granulomas formed afterhematogenous spread of pulmonary tuberculosis. Little is known aboutthe early pathogenesis of TBM and the role of Rich foci. We haveadapted the zebrafish model of Mycobacterium marinum infection(zebrafish–M. marinum model) to study TBM. First, we analyzedwhether TBM occurs in adult zebrafish and showed that intraperitonealinfection resulted in granuloma formation in the meninges in 20% ofthe cases, with occasional brain parenchyma involvement. In zebrafishembryos, bacterial infiltration and clustering of infected phagocyteswas observed after infection at three different inoculation sites:parenchyma, hindbrain ventricle and caudal vein. Infection via thebloodstream resulted in the formation of early granulomas in braintissue in 70% of the cases. In these zebrafish embryos, infiltrates werelocated in the proximity of blood vessels. Interestingly, no differenceswere observed when embryos were infected before or after earlyformation of the blood-brain barrier (BBB), indicating that bacteria areable to cross this barrier with relatively high efficiency. In agreementwith this observation, infected zebrafish larvae also showed infiltrationof the brain tissue. Upon infection of embryos with an M. marinumESX-1 mutant, only small clusters and scattered isolated phagocyteswith high bacterial loads were present in the brain tissue. Inconclusion, our adapted zebrafish–M. marinum infection model forstudying granuloma formation in the brain will allow for the detailedanalysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci andpotentially contribute to the development of better diagnostic tools andtherapeutics.

KEY WORDS: Tuberculous meningitis, Tuberculosis, Zebrafish,Mycobacterium marinum, Blood-brain barrier, ESX-1 mutant

INTRODUCTIONIn 1993, the World Health Organization (WHO) declaredtuberculosis (TB) a global public health emergency, with an

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1Department of Pediatric Infectious Diseases and Immunology, VU UniversityMedical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.2Department of Medical Microbiology and Infection Control, VU UniversityMedical Center, Van der Boechorststraat 7, 1081 BT, Amsterdam, TheNetherlands. 3Department of Pathobiology, Utrecht University, Faculty ofVeterinary Medicine, Yalelaan 1, 3508 TB, Utrecht, The Netherlands.

*Author for correspondence (a.vandersar@vumc.nl)

This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricteduse, distribution and reproduction in any medium provided that the original work is properlyattributed.

Received 3 January 2014; Accepted 10 June 2014

estimated 7- to 8-million cases and 1.3- to 1.6-million TB deaths peryear. By 2013, the situation had improved in many areas, butabsolute numbers remained virtually unchanged, with an estimated8.7-million new cases and 1.4-million TB deaths per year (WorldHealth Organization, 2013). Central nervous system (CNS)involvement, most commonly leading to tuberculous meningitis(TBM), is the most severe extra-pulmonary complication of TB andaccounts for ~1% of all TB cases (Wolzak et al., 2012). TBMespecially occurs in early childhood, with a peak incidence inchildren younger than 5 years (van Well et al., 2009), and has beenreported as the most common form of meningitis diagnosed inchildren in TB endemic areas (Rock et al., 2008). The increased riskof children to develop meningeal TB is presumably due to immatureinnate and adaptive immune responses (Sterling et al., 2007;Lewinsohn et al., 2004). This immaturity leads to a relative inabilityto contain primary infection in the lung, which increases the risk ofdisseminated disease (Sterling et al., 2007; Principi and Esposito,2012).

After pulmonary TB, tubercle bacilli can disseminate via thebloodstream and are able to cause infection at distant sites such ascervical lymph nodes (scrofula) or vertebrae (Pott’s disease). Ourunderstanding of the pathogenesis of TBM dates from the work ofRich and McCordock in the 1920s and 1930s. They were the first todescribe the theory that TBM arises from a caseating granuloma inthe brain instead of a direct consequence of a spread of tuberclebacilli to the meninges (Rich and McCordock, 1933). Otherresearchers, mainly pathologists, confirmed the Rich focus theory(Blacklock and Griffin, 1935; MacGregor and Green, 1937).However, it is not yet clear exactly what mechanisms lead to theformation of foci in the brain and its surrounding meninges.Different types of granulomatous lesions were described in all partsof the CNS, ranging from small and multiple caseous nodules tolarge exudative plaques (MacGregor and Green, 1937; Rich andThomas, 1946). A Rich focus can be latent, which causes the diseaseto remain enclosed for a long period. Alterations in immune statusand growth of the granuloma might lead to rupture and discharge ofbacteria into the subarachnoid space and meningitis can occur. Theinflammatory response to the discharged bacteria results ininflammatory exudation, mostly of the basal cisterns. This can befollowed by cranial nerve palsies, obliterative vasculitis, obstructionof cerebral spinal fluid (CSF) and formation of new granulomaselsewhere in the meninges or brain tissue (Rich and Thomas, 1946;Donald et al., 2005). Nowadays, the discussion about thepathogenesis of TBM development and the role of the Rich focus inpathogenesis still continues. A re-interpretation of the work of Richand McCordock might be required, because a relationship betweenmiliary TB and TBM cannot be completely excluded (Donald et al.,2005). Knowledge about early pathogenesis of TBM is important inthe search for early stage diagnostic tools, new treatment strategiesand vaccine development.

Modeling tuberculous meningitis in zebrafish usingMycobacterium marinumLisanne M. van Leeuwen1,2, Martijn van der Kuip1, Sameh A. Youssef3, Alain de Bruin3, Wilbert Bitter2, A. Marceline van Furth1 and Astrid M. van der Sar2,*

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Animal experiments have contributed to our knowledge of TBand TBM. A wide range of models to study TB and TBM exists, allwith their own benefits and disadvantages. Mice provide a goodmodel to study fundamental features of the immune response to TBand TBM and are used by different research groups (van Well et al.,2007; Be et al., 2008; Zucchi et al., 2012), but a major disadvantageof this model is the fact that mice form poorly organized granulomasafter infection with mycobacteria. Animals that do form well-structured granulomas with caseous necrosis are guinea pigs (Rich

and McCordock, 1933; Be et al., 2011), rabbits (Tsenova et al.,1998; Tsenova et al., 1999; Tsenova et al., 2002; Tsenova et al.,2006) and non-human primates (Young, 2009). The majordisadvantages of these models are the high costs, ethical problemsand the limited range of immunology reagents (Young, 2009).

The zebrafish (Danio rerio) is a small teleost fish with an innateand adaptive immune system (Lam et al., 2004; Meijer and Spaink,2011; Renshaw and Trede, 2012) and a blood-brain barrier (BBB), allof which are comparable to humans in both structure and function(Jeong et al., 2008; Xie et al., 2010; Fleming et al., 2013). The firstfeatures of the BBB, an endothelial cell layer connected by tightjunctions that forms the barrier between the blood and brain tissue(Abbott et al., 2006), are described to be present at 3 days post-fertilization (dpf) (Jeong et al., 2008; Xie et al., 2010), and maturationcontinues until 10 dpf (Fleming et al., 2013). Zebrafish are extensivelyused as a model organism; advantages include the fecundation andgrowth outside the mother, potential to study right from the single-cellembryonic stage, ease of genetic manipulation (Nasevicius and Ekker,2000) and the availability of a growing mutant library(http://www.sanger.ac.uk/Projects/D_rerio/zmp/). The transparency ofzebrafish larvae in combination with an increasing accessibility offluorescent tools (Ellett et al., 2011; Lawson and Weinstein, 2002;Meijer and Spaink, 2011; Tobin et al., 2012b) provides opportunitiesto study host-pathogen interaction in real time.

Mycobacterium marinum is one of the closest relatives ofmembers of the Mycobacterium tuberculosis complex and zebrafishinfected with this bacterium develop granulomas similar to those inhuman TB (Tobin and Ramakrishnan, 2008). It has been shown thatmycobacterium-macrophage interaction can initiate granulomaformation in the zebrafish (Davis et al., 2002; Ramakrishnan, 2013).Furthermore, M. marinum and M. tuberculosis share a lot ofimportant virulence factors, of which the ESX-1 locus is one of thebest examples. The mycobacterium ESX-1 locus, encoding a typeVII secretion system, plays a role in virulence (Abdallah et al., 2007;Simeone et al., 2012; Stoop et al., 2012) and is partially missing inthe vaccine strain Mycobacterium bovis Bacillus Calmette-Guérin(BCG) (Gordon et al., 1999). Virulent mycobacteria use the ESX-1locus to enhance macrophage recruitment and subsequentdissemination of disease (Volkman et al., 2004; Davis andRamakrishnan, 2009; Ramakrishnan, 2013). The ESX-1 system isrequired for phagosomal escape (van der Wel et al., 2007; Houbenet al., 2012; Simeone et al., 2012), which precedes host cell death.As such, infections with this mutant result in reduced cell death, areduced number of extracellular bacteria and an increased numberof bacteria per infected host cell (Gao et al., 2004).

In this study we set out to determine the optimal inoculation route[intraperitoneally (i.p.) versus intravenous (i.v.) versus direct CNSinjection] to induce TBM at different maturation stages of thezebrafish. We show that the zebrafish model of M. marinuminfection (zebrafish–M. marinum model) is an accessible andreproducible model to analyze the pathogenesis of early CNSgranuloma formation and the factors involved in this process. Inaddition, we show that the formation of the BBB does not influenceearly CNS granuloma formation.

RESULTSI.p. infected adult zebrafish develop granulomas inmeninges with brain involvementTo evaluate whether zebrafish can be used to study TBM we first re-examined sections of adult zebrafish acquired in previousexperiments (Appelmelk et al., 2008; Stoop et al., 2013) and lookedfor the presence of mycobacterial infections in the head regions,

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RESOURCE IMPACTBackgroundTuberculous meningitis (TBM), involving the central nervous system(CNS), represents the most severe extra-pulmonary complication oftuberculosis (TB). It is caused by infection with Mycobacteriumtuberculosis and affects, in particular, children below the age of 5 years.Early and rapid diagnosis of TBM is crucial for a favorable outcome;however, owing to non-specific early symptoms, diagnosis is oftendelayed. When disease progresses, symptoms become more prominentand include focal neurological signs and convulsions, which might lead,in the final stage, to irreversible neurological damage or even death. Aclear neuropathological feature of TBM is the formation of granulomas(i.e. collections of immune cells) in the brain tissue or meninges. Thesepathological structures are the so-called Rich foci, which form after thespread of pulmonary TB via the bloodstream. Little is known about themechanisms of Rich foci formation and their role in the earlypathogenesis of TBM. Therefore, it is necessary to improve knowledgeof the initial stages of meningitis development, which might contribute tothe design of early stage diagnostic tools as well as of novel therapeuticapproaches and vaccines.

ResultsIn this study, the authors adapted the zebrafish model of M. marinuminfection (zebrafish–M. marinum model) to investigate in more detail theearly pathogenesis of TBM. They first confirmed that M. marinum, aclose relative of Mycobacterium tuberculosis, could successfully infectadult zebrafish, which have a fully developed immune system and blood-brain barrier, and that there was granuloma formation in the meninges ina minority of cases (as is the case in humans in endemic areas).Subsequently, they characterized the initial phases of zebrafish infectionby using different routes of bacteria inoculation at early developmentalstages; in most of the zebrafish embryos (which only have innateimmunity) both local and systemic injections caused abundant braininfection, with formation of bacterial clusters that were identified as earlygranulomas. All clusters contained both mycobacteria and a populationof either epithelioid or foamy macrophages, but their development wasnot influenced by the presence of the blood-brain barrier. By contrast,infection with the mycobacteria mutant lacking the ESX-1 secretionsystem (which is involved in virulence factor secretion) resulted in theformation of smaller clusters with a high number of phagocytosedbacteria, but did not seem to affect migration of bacteria from thebloodstream to the brain tissue.

Implications and future directionsThis study shows that the zebrafish–M. marinum model is particularlysuitable for characterization of the early steps in the formation of braingranulomas, their immunological composition and the effect of bacterialvirulence factors in the context of TBM. This knowledge is of fundamentalimportance, particularly considering that the CNS is poorly investigatedin the field of tuberculosis research. The advantages of the zebrafishmodel include its small size, the ease of breeding and of geneticmanipulation, as well as the great similarities with the human immunesystem and blood-brain barrier. Moreover, a unique feature of this modelis the transparency of the zebrafish embryos, which, in combination withfluorescent tools, could allow real-time imaging of host-pathogeninteractions in the study of infectious diseases, including TBM. Thisresearch approach could potentially extend our knowledge of bacterialvirulence factors and host characteristics, which will help to advance bothearly diagnosis and treatment of disease.

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especially the brain parenchyma or the meninges. These zebrafishwere i.p. infected with M. marinum at 1 year of age, sacrificed at 8weeks (±6 days) post infection (wpi) and all fish showed granulomaformation in the abdominal organs. In five out of 26 fish, formationof granulomas (range of one to six granulomas per zebrafish) wasalso found in close relationship with the brain and meninges.Granulomas mostly affected the meninges and submeningeal space,were multifocal to coalescing, variably sized (50-300 μm indiameter) and fairly circumscribed (Fig. 1A). The brain parenchymaltissue beneath the meningeal granulomas showed minimallymphocytic inflammation and gliosis, but clear infection of theparenchyma was not observed. The granulomas were composed ofa uniform population of epithelioid and foamy macrophages rarelyaccompanied by the presence of lymphocytes (Fig. 1B, blackarrow). Although no clear caseation, calcification or fibrosis wasobserved, individual macrophages at the center of granulomasexhibited cellular degeneration and necrosis as a first sign ofgranuloma maturation (Fig. 1B, arrowheads). Ziehl-Neelsen (ZN)staining confirmed the presence of mycobacteria in the cytoplasmof macrophages (Fig. 1C). Interestingly, severe congestion of themeningeal and brain parenchymal blood vessels was present aroundgranulomas. (Fig. 1A,B, yellow arrow). Furthermore, two fish hadorbital granuloma formation (supplementary material Fig. S1).Because orbital TB in humans is either a result of direct extensionfrom a tubercular focus in the paranasal sinuses or by hematogenousspread from a distant granuloma, it should not be considered to bethe same as CNS TB (Madge et al., 2008). To summarize, in thepresence of a full-grown immune system, zebrafish developgranulomas in close relation with brain tissue and meninges after i.p.infection with M. marinum. Thereby, we demonstrate that thezebrafish–M. marinum infection model is a representative andnatural model to study TBM pathogenesis. Interestingly, only aminority of adult fish present infection in the CNS. This iscomparable to the human situation in TB endemic areas, whereTBM is rare in adults as compared with children (Rock et al., 2008;van Well et al., 2009).

Zebrafish embryos show abundant early granulomaformation in the brain tissueLocal infectionTo study the effect of M. marinum infection in brain tissue, wedirectly inoculated M. marinum into the brain parenchyma orhindbrain ventricle of zebrafish embryos (Fig. 2A; supplementarymaterial Table S1). In 95-100% of all cases this resulted in theformation of bacterial clusters in the targeted brain area (Fig. 2B).

Depending on the amount of infection in the brain, infectedzebrafish were divided into three groups. Zebrafish were labeled ashaving large clusters when 50% or more of the brain area containedred fluorescent bacteria, medium clusters when 10-50% was infectedand small clusters when less than 10% was infected (Fig. 2C-E).After direct parenchymal infection, large clusters were found in 60%of the cases and only a minority of zebrafish contained smallclusters. The clusters formed after hindbrain ventricle infection weremostly medium sized, but large and small clusters were detected aswell (Fig. 2F). To obtain more precise information about thelocalization of these early granulomas, the embryos werehistologically analyzed using anti-acetylated tubulin staining of thenerve tracts (Fig. 2G-I; supplementary material Table S1). Allinfected embryos showed early granuloma formation at the injectionsite as well as disseminated disease, defined by single bacteria andearly granulomas at a distant location (in both parenchyma andventricular system). Expansion of the primary cluster afterparenchymal infection usually took place in the direction of theparenchyma. Interestingly, the bacteria seemed to spread more easilyvia the cerebral spinal fluid (CSF) in the ventricular system, asexemplified by clusters in the ventricle wall (Fig. 2H, arrow).Similarly, clusters in the ventricle wall were found after hindbrainventricle infection and displayed clear growth into the parenchyma,indicating that dissemination of bacteria can occur throughout theentire brain after local inoculation of bacteria into the parenchymaand the hindbrain ventricle.

Systemic infectionA more natural route for infection of brain tissue is probably via theblood circulation. Therefore, we utilized caudal vein injection asmodel for hematogenous spread of disease. All 135 examinedembryos contained disseminated infection and, of these, 70%(94/135) displayed an infection in the brain area. The amount ofembryos with infection in brain tissue seemed to be associated withthe number of colony forming units (CFU) used for the systemicinjection, i.e. the percentage of zebrafish embryos with braininfection was the lowest (41%) in the experiment with the lowestnumber of CFU (117 CFU) injected in the caudal vein. In contrastto local brain infection, i.v. infection resulted predominantly in smalland medium bacterial clusters in the brain area, whereas largeclusters were less commonly seen (Fig. 2F).

Anti-acetylated tubulin staining revealed that the clusters wereindeed formed in the brain parenchyma or ventricular system of thezebrafish embryo (Fig. 2J; supplementary material Table S1). Mostof the embryos contained more than one cluster and clusters were

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Fig. 1. Granulomas in adult zebrafish after intraperitoneal infection. (A) Coronal section of adult zebrafish at 50 days after infection with M. marinum E11.Multifocal to coalescing granulomas (black arrows) affecting the meninges and submeningeal space structure can be seen, the brain parenchymal tissue (*)beneath the granulomas shows minimal lymphocytic inflammation, and the regional meningeal blood vessels are markedly congested (yellow arrow). Scalebar: 100 μm. Granuloma is enlarged in panels B and C. (B) Granulomas are composed of epithelioid and foamy macrophages (black arrow) that occasionallyexhibited degeneration and necrosis (arrowheads). Scale bar: 20 μm. (C) Same granuloma as shown in panel B stained with ZN. Multiple acid-fast bacilli arepresent in the cytoplasm of the macrophages (black arrows). Scale bar: 20 μm.

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found to form in every part of the brain. Histopathological analysis[hematoxylin and eosin (HE) staining] confirmed this finding, andshowed that meningeal granulomas occasionally impinged on theunderlying brain parenchyma causing brain compression and severebrain tissue loss (Fig. 3). To examine the relation between clusterformation and vascular patterns in more detail, Tg(Fli1:GFP)y1

casper zebrafish embryos were used. Infection of these embryosshowed that both clustered and single mycobacteria were found inthe parenchyma, often closely located to blood vessels (Fig. 2K,L;supplementary material Table S1), indicating that mycobacteriamigrate out of blood vessels and form clusters in the brain tissue.

Granuloma compositionTo confirm whether the observed bacterial clusters were actually earlygranulomas containing phagocytic cells, an anti-L-plastin staining wasperformed. All infection routes showed formation of clusters in whichmycobacteria and phagocytes colocalized (Fig. 4; supplementarymaterial Table S2). The phagocyte-mycobacterium area ratio (inconfocal images) was equal in ~80% of the observed clusters(supplementary material Table S2). In the remaining clusters, the areacovered by mycobacteria was higher compared with the area coveredby phagocytes and these clusters were surrounded by many non-

infected phagocytes in most cases (Fig. 4F). Interestingly, differencesin intensity of L-plastin were observed between different phagocytesinside and around early granulomas (Fig. 4B,D-F, arrows). In additionto confocal microscopy, a more detailed histopathological analysis(HE and ZN staining) of embryos showed that granulomas werecomposed of a uniform population of either epithelioid or foamymacrophages (Fig. 3A,B,D,E, black arrows) rarely containingheterophils (Fig. 3B, arrowhead), the zebrafish neutrophil counterpart,and contained many acid-fast bacilli (Fig. 3C,F, black arrow). Nonecrosis or fibrosis was found, fitting with the idea that these are earlygranulomas. Besides cluster formation, solitary bacterial spreadthrough the brain was observed in all embryos (based on confocalmicroscopy). These single bacteria were mostly located near a clusterand observed both inside and outside phagocytes (Fig. 4).

Presence of the BBB does not influence early granulomaformation in the brain parenchymaIn zebrafish embryos, the BBB is largely formed at 3 dpf, preventinglarge particles from migrating into the brain parenchyma (Jeong etal., 2008; Xie et al., 2010; Fleming et al., 2013). We confirmed thepresence and functionality of this early barrier in our casper fishpopulation (supplementary material Fig. S2) and investigated

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Fig. 2. Three infection routes compared in the embryo model. (A) Photograph of a casper embryo at 2 dpf. Red arrows indicate the three different infectionroutes used. (B) Casper embryo at 7 dpf. The red line indicates the brain area. (C) Example of a small cluster. Left panel shows bright-field image; right panelshows corresponding fluorescent image. (D) Example of a medium cluster. (E) Example of a large cluster. (F) At 5 dpi, the infection was analyzed withfluorescence microscopy. Fluorescent bacterial clusters in the brain were counted visually. The clusters were scored, depending on their size, as small, medium orlarge. CV, caudal vein; Par, parenchyma; HV, hindbrain ventricle. (G-J) Z-stack of zebrafish embryos, at 7 dpf, stained with anti-acetylated tubulin (labelling axons;green) and infected with M. marinum E11 (red). Scale bars: 150 μm. (G) Anatomy of the zebrafish brain: FB, forebrain; MB, midbrain; HB, hindbrain; OT, optictectum (L, left; R, right); OB, olfactory bulb; FV, forebrain ventricle; TV, tectal ventricle; HV, hindbrain ventricle; A, aquaductus; E, eye. (H) Embryo with multipleclusters after infection into the parenchyma. Arrow indicates a cluster in the ventricular wall. (I) Embryo infected into the hindbrain ventricle, with a cluster in theventricle. Marked area indicates the hindbrain ventricle. (J) Embryo infected via the caudal vein, with a cluster in the parenchyma. (K,L) Tg(Fli1:GFP)y1 casperembryo (Fli1:GFP in green) at 5 days after infection via the caudal vein with M. marinum E11 (red). Both panels are single Z-slices and show the relationshipbetween mycobacteria and blood vessels in the brain, which indicates migration of mycobacteria from bloodstream to brain tissue. Scale bars: 35 μm.

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whether this barrier influences early granuloma formation in thebrain. Therefore, embryos were infected i.v. before (2 dpf) and after(4 dpf) the start of BBB formation. The number of zebrafish withinfection in the brain, as a percentage of the total number of infectedzebrafish, ranged from 41 to 73% for zebrafish infected before BBBdevelopment, versus 52-83% after BBB development (Fig. 5A,B).The size of the formed clusters of both groups was equal, i.e. smallclusters were mostly observed, medium clusters were less commonand large clusters were only seen once or twice (Fig. 5C). Theseresults show that early granulomas can be formed in the brain in thepresence of the early BBB and that the presence of this barrier doesnot seem to influence the amount of infection.

To confirm that even a fully matured BBB does not influence theamount of infection in the CNS after i.v. injection, a group of larvae(21-25 dpf) was infected via the bloodstream. Thirteen out of 71(18%) infected larvae showed bacterial clusters in the brain area.Interestingly, six of these 13 larvae only showed infection in thebrain area. With histochemical analysis using an anti-L-plastinstaining and an anti-acetylated tubulin staining, we confirmed thatthese clusters were actually early granulomas consisting ofphagocytic cells and mycobacteria, and were located in the brainparenchyma or ventricles (Fig. 5D-H). The percentage of infectionin the larvae brain (21 dpf) was reduced as compared with theembryos (2 or 4 dpf), so further maturation of the BBB might reduceor delay migration of mycobacteria. However, BBB maturation doesnot seem to block migration, which leads to the assumption that,even in the presence of a fully developed BBB and a partiallydeveloped adaptive immune system, bacterial infiltration and clusterformation in the brain occurs efficiently.

Mycobacteria with a defective ESX-1 secretion system showefficient colonization of brain tissueHere, using the eccCb1::tn mutant, a bacterial M. marinum strainwith a disruption in the ESX-1 locus, we examined whether the

ESX-1 locus is required for invading the CNS. First, we directlyinjected the eccCb1::tn mutant bacteria into the brain parenchymaor hindbrain ventricle. Similar to what was shown in previousstudies (Volkman et al., 2004; Davis and Ramakrishnan, 2009;Stoop et al., 2011), we observed significant differences between thewild-type and the eccCb1::tn mutant in cluster formation and clustersize (Table 1; supplementary material Tables S1-S4). In mostembryos infected with the eccCb1::tn mutant, we only detectedsmall early granulomas (compare Fig. 6A,B). This differencebetween the early granulomas formed by the eccCb1::tn mutant orwild-type mycobacteria is clear upon measuring the diameters(Fig. 6C). Furthermore, in the eccCb1::tn mutant group, weobserved numerous isolated phagocytes filled with many bacteria inall embryos (Table 1; Fig. 6D). These highly infected phagocytesdid not form early granulomas and were scattered throughout theentire brain. In contrast, we never found more than four solitaryphagocytes containing mycobacteria in wild-type-infected embryos.Subsequently, we tested the effect on CNS invasion after i.v.infection of the ESX-1 mutant. Again, in these embryos the highlyinfected extragranulomatous phagocytes seemed to dominate andsporadic early granulomas were only found in half of the embryos(Fig. 6E; supplementary material Tables S3, S4). Importantly, 43%of the eccCb1::tn-mutant-injected embryos contained infection inthe brain area, indicating that CNS infiltration was not blocked.

Closer analysis of M. marinum eccCb1::tn-infected Tg(Fli1:GFP)y1

casper embryos, stained with L-plastin, revealed that phagocytescontaining mycobacteria were located both inside and outside bloodvessels (Fig. 6F-I). This group of ten embryos contained 37 infectedphagocytes of which 12 were found inside a blood vessel and 25 werefound in the brain tissue (supplementary material Table S2).

In conclusion, infection of zebrafish embryos with the ESX-1mutant resulted in the formation of smaller clusters and a highernumber of phagocytes filled with an abundant load of bacteria, asexpected based on earlier publications (Volkman et al., 2004; Davis

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Fig. 3. Histopathological analysis of granulomas in the brain of a zebrafish embryo. (A) Coronal section of an embryo (7 dpf; 5 days after infection withM. marinum E11) showing an early granuloma (black arrows) attached to the meninges (yellow arrow), causing impingement of the underlying brainparenchyma (*). Scale bar: 50 μm. Marked area is enlarged in panels B and C. (B) The early granuloma is composed of epithelioid macrophages (black arrow)and heterophils (arrowhead). (C) ZN stain of granuloma depicted in panel B showing the presence of acid-fast bacilli (black arrow). Scale bars: 20 μm.(D) Coronal section of an embryo (7 dpf; 5 days after infection with M. marinum E11) showing an early granuloma (black arrow) present in the submeningealspace causing severe impingement of the underlying brain parenchyma (*). Scale bar: 50 μm. Marked area is enlarged in panels E and F. (E) The earlygranuloma is composed of foamy macrophages (black arrow) and (F) shows moderate numbers of acid-fast bacilli (black arrow). Scale bars: 20 μm.

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and Ramakrishnan, 2009). In addition, our experiments indicate thatbacterial migration from bloodstream to brain parenchyma stilloccurs when almost no extracellular bacteria are present.

DISCUSSIONIn this paper we adapted the zebrafish–M. marinum infection modelto study the early pathogenesis of TBM. From neuropathologystudies, it is well known that TBM always starts with granulomaformation in brain tissue or meninges, and our model has thepotential to unravel the first steps in the establishment of thesegranulomas. We show that infection of zebrafish embryos (withinnate immunity only) and larvae (with both innate and partialadaptive immunity) via different inoculation routes led to theformation of early granulomas in the brain parenchyma and in theventricular systems. In addition to the embryo and larval model,granulomas were also observed in the CNS of adult zebrafish afteri.p. infection with M. marinum. An interpretation of our findings isschematically depicted in Fig. 7.

Model developmentCompared with the adult zebrafish, the embryo and larval TBMmodel is more flexible and can be tuned depending on the researchquestions. In zebrafish embryos, differences in cluster size wereobserved between the three different infection routes. Interestingly,infection via the caudal vein mainly led to the formation of smalland medium early granulomas in the brain. Cluster size as well asthe number of zebrafish with infections in the brain area seemedto depend on the infection dose, suggesting that severity of TBMcorrelates with the bacterial load in the blood. The Rich focus

theory describes that an early granuloma is formed afterhematogenous spread and that TBM is not a direct consequence ofmiliary TB. In contrast, our results suggest that there might be adirect correlation between higher bacterial load in the blood andhigher risk for TBM and higher numbers of granulomas formed inthe brain. This is in line with the idea that the Rich focus theoryneeds to be reconsidered (Donald et al., 2005). An alternativetheory is that bacteria spread via the CSF circulation in theventricular system, which leads to the formation of granulomatousstructures or to direct infection of the meninges. Our data showsabundant infection in the ventricular system, which suggests thatentrance of mycobacteria into the brain parenchyma via theventricular wall is a possibility.

Composition of granulomas in the brainThe major cell types present in early granulomas in embryos andmore matured granulomas in adult fish are epithelioid or foamymacrophages. With this histopathological data, it is difficult todetermine whether these macrophages have a systemic origin orwhether they are derived from microglia (specific brain phagocytes).Interestingly, with the immunohistochemical staining of embryos forthe common leukocyte marker L-plastin, we observed differences inL-plastin intensity between the different phagocytes. It is describedthat macrophages that have colonized the brain and retina undergoa phenotypic transition between 2 and 3 dpf. The expression of L-plastin is downregulated and macrophages start to express highlevels of apolipoprotein-E (Herbomel et al., 2001; Meijer andSpaink, 2011). We hypothesize that this downregulation couldexplain the differences we observed. All granulomas contained

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Fig. 4. Composition of early granulomas. (A) Z-stack of the head of a zebrafish embryo (7 dpf) stained with anti-L-plastin, labeling phagocytic cells (green),infected with M. marinum E11 (red) via the caudal vein (5 dpi). Marked area is enlarged in panels B-E. (B-D) Z-stack of an early granuloma, with phagocyticcells stained with anti-L-plastin (B), M. marinum E11::mCherry (C) and merge (D). A phagocyte with high L-plastin intensity (closed arrow) and a phagocytewith low L-plastin intensity (open arrow) are depicted in panels B and D. (E) Single Z-slices of granuloma shows colocalization of phagocytes andmycobacteria. Arrows indicate different intensities of L-plastin, eliminating the possible effect of overlapping phagocytes in the Z-stack in panel B and D. (F) Z-stack of an early granuloma containing relatively more mycobacteria than phagocytes. Lots of phagocytes surrounding the cluster are probably still migrating tothe cluster. The parenchymal infection route was used. Images were taken at 5 dpi. Scale bars: 150 μm (A), 35 μm (B-F).

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phagocytes with high and low L-plastin intensity, which might bean indication that granulomas in the brain consist of microglia andmacrophages or neutrophils with a systemic origin. Although we didnot find clear caseation of granulomas in brain tissue in our adultfish, signs of maturation, such as the presence of lymphocytes andcentral necrosis of macrophages, were observed. The similarities incomposition between granulomas in embryos and adult fish suggestthat embryonal clusters are indeed precursors of adult granulomas.Taking these data together, this model provides opportunities to

study the composition and behavior of brain granulomas in an innateversus adaptive setting.

Bacterial migrationWe showed that, after infection via the bloodstream, earlygranulomas were formed in the brain parenchyma and theventricular system of both embryos and in larvae containing a fullydeveloped BBB. Our experiments with Tg(Fli1:GFP)y1 casperembryos confirmed these findings and indicated that mycobacteria

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Fig. 5. Presence of the blood-brain barrier. (A,B) Number of zebrafish embryos with bacteria in the brain area. Embryos were infected either before earlyBBB development (2 dpf) or after early BBB development (4 dpf) (four groups of embryos were used for each time point). No significant differences were found;P=0.3263. (C) Distribution of cluster size is shown as percentages of the total number of infected zebrafish. (D) Casper larvae (32 dpf) microinjected at 21 dpfwith M. marinum E11 (red) in the heart. Analysis was performed at 11 dpi with anti-L-plastin staining (green). Marked area is enlarged in panels E-H. Scale bar:300 μm. (E-G) Z-stack of cluster with a lot of phagocytic cells (single channel not shown), mycobacteria (E) and merge of both channels (F). Scale bars: 60 μm.(G,H) Single Z-slices at different Z-levels show colocalization of phagocytes and mycobacteria and cluster formation. Scale bar: 30 μm.

Table 1. Role of the ESX-1 locus in the formation of early brain granulomasTotal no. single phagocytes

Embryos Total no. granulomas filled with bacteria

Parenchyma: M. marinum E11 23 67 15Hindbrain ventricle: M. marinum E11 24 86 26Caudal vein: M. marinum E11 25 86 43Parenchyma: M. marinum eccCb1::tn 23 81 173Hindbrain ventricle: M. marinum eccCb1::tn 20 60 123Caudal vein: M. marinum eccCb1::tn 25 16 131

Overview of total number of granulomas and total number of single phagocytes filled with bacteria, counted in all embryos stained with anti-acetylated tubulinand anti-L-plastin. In all eccCb1::tn-infected embryos, phagocytes filled with a large bacterial load were found; in E11-infected embryos this was not the case.Parenchyma (Par) E11 versus Par ESX-1 mutant: P<0.0001, hindbrain ventricle (HBV) E11 versus HBV ESX-1 mutant: P<0.0001, caudal vein (CV) E11 versusCV ESX-1 mutant: P=0.0135. See supplementary material Tables S2, S3. D

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leave the bloodstream and are able to establish new granulomas insurrounding tissue; because individuals with an intact BBB dodevelop TBM, it is clear that mycobacteria traverse this barrier.However, the exact mechanism is not yet known.

The BBB consists of endothelial cells with tight junctions,surrounded by a continuous basement membrane and astroglial end-feet (Abbott et al., 2006), which limits the exchange of pathogens,pharmacological compounds, immune cells and mediators(Ransohoff et al., 2003). Three major mechanisms of traversalacross this BBB are described for other causative pathogens ofmeningitis: transcellular migration, paracellular migration or theTrojan-horse mechanism whereby the pathogen uses a macrophageas carrier. Streptococcus pneumoniae, Haemophilus influenzae andNeisseria meningitides, for example, use the transcellularmechanism by binding to the endothelium of the BBB using lamininreceptors and by expressing phosphorylcholine, which mimicsplatelet-activating factor (Orihuela et al., 2009). Cryptococcusneoformans, the most common cause of fungal meningitis, hasshown to use both the transcellular and the Trojan-horse mechanism.

Liu et al. (Liu et al., 2013) described the dependence of traversalacross the BBB on inositol (Liu et al., 2013), of which highconcentrations are found in human and animal brains, especiallyaround the BBB. Mycobacteria do have an extensive inositolmetabolism, which leads to the hypothesis that host inositol mightplay an important role in mycobacterial migration over the BBB aswell (Morita et al., 2011).

Because mycobacteria are known to be intracellular pathogens,the Trojan-horse mechanism is a plausible hypothesis for traversalacross the BBB. In vitro studies with a bilayer of alveolar epithelialcells and human lung endothelial cells have strengthened thishypothesis by showing that M. tuberculosis-infected monocytescross the alveolar wall with greater efficiency than uninfectedmonocytes or mycobacteria alone (Bermudez et al., 2002).Furthermore, invasion of alveolar epithelial cells by mycobacteriaenhances this translocation by inducing chemokine release(Bermudez et al., 2002), which suggests that other traversal routesmight play an important role as well. The BBB is distinct from thealveolar wall, but the mechanism that mycobacteria and infected

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Fig. 6. Role of the ESX-1 locus in the formation of early brain granulomas. (A) Example of a compact cluster with phagocytic cells and bacteria afterinfection into the parenchyma with M. marinum E11 (red), stained with anti-L-plastin (green). (B) Example of cluster after infection into the parenchyma with M.marinum eccCb1::tn (red), stained with anti-L-plastin (green). (C) Overview of diameter of individual granulomas counted in embryos infected via parenchyma(Par), hindbrain ventricle (HBV) or caudal vein (CV), with either M. marinum E11 or the isogenic eccCb1::tn mutant. The number of granulomas is described inTable 1. Par E11 versus Par F3.1: *P<0.0001, HBV E11 versus HBV F3.1: **P<0.0001, CV E11 versus CV F3.1: ***P=0.0135. (D) Example of single phagocyticcells filled with mycobacteria in an embryo infected into the parenchyma with eccCb1::tn mutant (red), stained with anti-L-plastin (green). (E) Example of singlephagocytic cells filled with mycobacteria in an embryo infected via the caudal vein with eccCb1::tn mutant (red), stained with anti-L-plastin (green). Scale bars:35 μm (A,B,D,E). (F-I) Z-stack of the relationship between vasculature, phagocytes and bacteria. Caudal vein infection with M. marinum eccCb1::tn mutant,with (F) Tg(Fli1:GFP)y1 casper embryo showing blood vessels, (G) L-plastin Alexa-Fluor-633 showing phagocytic cells and (H) eccCb1::mcherry showingbacteria. (I) Merge of panels F-H. Closed arrow indicates a phagocyte containing mycobacteria inside a blood vessel; open arrow indicates a phagocytecontaining mycobacteria outside a blood vessel; arrowhead shows a bacterial cluster probably inside a dying phagocyte. Scale bars: 50 μm (F-I).

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macrophages use to cross this barrier might be comparable to whatthey use in the brain. However, with the current in vivo models forTBM, it has never been shown how mycobacteria actually leave thebloodstream and enter the brain. Our adapted zebrafish–M. marinummodel for TBM is the first in which these mechanisms can bestudied.

Zebrafish embryos infected with the ESX-1 mutant containedmany individual phagocytes filled with mycobacteria scatteredthroughout the brain, much like the highly infected individualmacrophages scattered throughout the tissue reported by Volkmanet al. (Volkman et al., 2004). Interestingly, migration ofmycobacteria from the bloodstream to the brain parenchyma andgranuloma formation in the brain is therefore not dependent on anintact ESX-1 locus. Attenuated M. marinum ESX-1 mutantsreplicate in the phagolysosome and cannot translocate to the cytosol(van der Wel et al., 2007; Houben et al., 2012). In addition, celldeath does not occur as quickly as in wild-type infection and, as aconsequence, a lower number of extracellular bacteria are present(Gao et al., 2004; Houben et al., 2012). Together, our results indicatethat traversal over the BBB is still possible in a setting with lowlevels of extracellular bacteria, which leads to the hypothesis thatmycobacteria possibly make use of host cells, i.e. macrophages, tomigrate out of the bloodstream.

Clinical implicationKnowledge about the morphological characteristics of granulomas,reflected by systemic and tissue-specific immune responses andmycobacterial virulence factors, is important to improve diagnosticand therapeutic strategies. Histopathological differences in types ofgranulomas in patients with TBM have been described since theearliest histopathological studies of Rich and McCordock, but alsoin a recent human postmortem study by our group (D. Zaharie andA.M.v.F., unpublished). In our zebrafish model, we have shown thatboth embryos and adults can be used to study granulomacomposition in great detail, and we observed considerablesimilarities with human neuropathology. With these two modelsystems (embryo versus adult), the importance of differentgranuloma types and influences of innate versus adaptive immunitycan be studied in a larger extent. This can subsequently be correlatedto inter-individual genetic variability of the innate and/or adaptiveimmune responses.

A striking finding in our histopathological data of adult zebrafishwas the presence of a large amount of congested vessels aroundmeningeal granulomas. In humans, a serious complication of TBMis the development of obliterative vasculitis and subsequentinfarction (Rock et al., 2008). Interestingly, this means that thisfeature of TBM pathogenesis can also be studied in the zebrafish.

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Fig. 7. Graphical abstract: schematic overview of our findings.(A) Schematic overview of our findings in experiments with M.marinum E11. Injection of mycobacteria in the parenchyma (circled‘A’) or into the hindbrain ventricle (circled ‘B’) leads to uptake bymacrophages (1 and 2, respectively) and formation of an earlygranuloma in the brain parenchyma (3) or the ventricular system (4),respectively. Bloodstream infection (circled ‘C’) also leads to infectionof macrophages and cluster formation in the brain parenchyma (5) orthe ventricular system (6) by an unknown mechanism (?). Isolatedbacteria and infected macrophages can disseminate throughout thebrain tissue (7) and can invade the parenchyma via the ventricularsystem as well (8). (B) Schematic overview of our findings inexperiments with M. marinum EccCb1::tn. Injection of mycobacteriain the parenchyma (circled ‘A’) or into the hindbrain ventricle (circled‘B’) leads to uptake by macrophages (1 and 2, respectively) andformation of a small early granuloma in the brain parenchyma (3) orthe ventricular system (5), respectively. In addition, isolated highlyinfected macrophages can be found in the parenchyma (4) or theventricles (6) in response to injection. Bloodstream infection (circled‘C’) also leads to infection of macrophages and in some cases tocluster formation in the brain parenchyma (7) or the ventricularsystem (8) by an unknown mechanism (?). Hypothetically,mycobacteria are transported out of the bloodstream bymacrophages in a Trojan-horse mechanism. Isolated bacteria andinfected macrophages can invade the parenchyma via the ventricularsystem as well (9). See supplementary material Table S1-S4.

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In relation to histopathological characteristics of granulomas,susceptibility to infection is another subject of interest. Increasedsusceptibility to both pulmonary and meningeal TB infection wasalready shown to be influenced by the Toll-like receptor pathway(Hawn et al., 2006). Recently, inter-individual differences in TNF-α response of zebrafish and humans have been studied (Tobin et al.,2010). TNF-α regulates activation of macrophages, recruitment ofother inflammatory cells, induction of cytokine/chemokineproduction and the induction of apoptosis. Therefore, TNF-α playsa key role in granuloma development. Humans treated with TNF-αneutralizing drugs show increased incidence of TB reactivation(Keane et al., 2001). The protein leukotriene A4 hydrolase, encodedby LTA4H, regulates the balance between pro- and anti-inflammatory responses. Individuals with a mutation in both allelesof the LTA4H gene develop an anti-inflammatory response with littleTNF-α production, whereas individuals having two wild-type alleleshave a pro-inflammatory phenotype with abundant TNF-α levels(Tobin et al., 2010; Tobin et al., 2012a). Both scenarios aredetrimental to the host, leading to bacterial overgrowth andhyperinflammation, respectively. On the other hand, intermediateTNF-α levels, as seen in individuals who are heterozygous forLTA4H, result in moderate inflammation, controlled infection andlow risk of severe disease or death (Tobin et al., 2010; Tobin et al.,2012a). It is known that corticosteroids are overall beneficial inTBM outcome (Prasad and Singh, 2008), but thalidomide (a TNF-α inhibitor) seems to work only in a subset of cases (Schoeman etal., 2004; Schoeman et al., 2006). Especially the TB abscess of theCNS is noted to be less responsive to conventional treatment, butgood results are reported to anti-TNF therapy in these cases(Schoeman et al., 2006). Thus, from a clinical point of view,knowledge of baseline response levels of TNF-α and subsequentgranuloma morphology might have therapeutical consequences indeciding whether to start with inhibiting immunomodulatory drugs,such as corticosteroids or thalidomide. In our model, differences ingranuloma number, size and ratio of phagocytes and mycobacteriawere observed as well. Also, we showed that mycobacterialvirulence factors had a clear impact on granuloma formation andstructure. Therefore, this model is an excellent tool to study theformation and behavior of different types of granulomas.

ConclusionIn conclusion, we have established a reproducible model to studythe pathogenesis of TBM in the zebrafish. Our model focusedespecially on the brain, an organ system that is often neglected. Themodel can be used for different research questions, and providesopportunities to further extend our knowledge about both bacterialvirulence factors that influence granuloma formation and hostcharacteristics leading to differences in type of granuloma and earlydisease outcome.

MATERIALS AND METHODSBacterial strains, growth conditions and injection stocksTwo different wild-type strains of M. marinum were used in this study: thehuman isolate, M. marinum M strain, originally isolated from humanpatients with fish tank granulomas (Ramakrishnan and Falkow, 1994), andthe sea bass isolate M. marinum E11 (van der Sar et al., 2004). In addition,we used the eccCb1::tn mutant of E11, which is known to be defective forESX-1 secretion (Stoop et al., 2011). All M. marinum strains were grown at30°C in Middlebrook 7H9 broth (Difco) with 10% Middlebrook albumin-dextrose-catalase (ADC; BD Bioscience) and 0.05% Tween-80 or onMiddlebrook 7H10 agar (Difco) supplemented with 10% oleic-acid-albumin-dextrose-catalase (OADC; BD Bioscience). pSMT3-DsRed andpSMT3-mcherry were electroporated into the M. marinum E11 strain, M

strain and eccCb1::tn (Stoop et al., 2011), in order to be able to visualizebacteria during infection in zebrafish embryos. Transformants of M.marinum M strain, E11 and eccCb1::tn were selected on plates containing50 μg/ml hygromycin. Injection stocks were prepared by growing bacteriauntil the logarithmic phase. Bacteria were washed with 0.3% Tween-80 inphosphate buffered saline (PBS) to declump the bacteria, resuspended inPBS with 20% glycerol and stored at −80°C. Before use, bacteria wereresuspended in PBS containing 0.17% (V/V) phenol red (Sigma) to aidvisualization of the injection process.

Animals and injection procedureMaintenance of adult casper zebrafish (White et al., 2008) and adultTg(Fli1:GFP)y1 casper zebrafish (Lawson and Weinstein, 2002) took place at26°C in aerated 5 liter tanks, in a 10:14 hour light:dark cycle. The eggs werecollected within the first hour post-fertilization (hpf) and kept at 28°C. At 48hpf, embryos were mechanically dechorionated and infected by microinjectionin the caudal vein, the hindbrain ventricle or the brain parenchyma (Fig. 2A).Injection was performed as described previously (Benard et al., 2012). At 4days post-infection (dpi) (for M. marinum M) or 5 dpi (in the case of M.marinum E11 and M. marinum eccCb1::tn), bacterial infection was monitoredwith a Leica MZ16FA fluorescence microscope. Bright-field and fluorescenceimages were generated with a Leica DC500 (DFC420C) camera and earlygranuloma formation was analyzed visually and quantified with custom-madesoftware (Stoop et al., 2011) (for additional information, see http://bio-imaging.liacs.nl/galleries/granulomaload/). Larvae were microinjected in theheart at 21-25 dpf, and bacterial infection was monitored at 11 dpi. Followinganalysis, embryos and larvae were fixed overnight in 4% (V/V)paraformaldehyde (EMS, 100122) dissolved in PBS, and stored in 100%methanol at −20°C for immunohistochemical staining and confocal imaging.To determine the exact number of bacteria injected, the injection volume wasalso plated on 7H10 plates. During injection and microscopic examining,embryos and larvae were anesthetized in egg water with 0.02% (W/V)buffered 3-aminobenzoic acid (Tricaine; Sigma-Aldrich, A-5040). Allprocedures involving zebrafish embryos and larvae were performed incompliance with local animal welfare laws. The number of injected embryosand larvae are listed in supplementary material Fig. S3.

BBB functionality assay with FD4Fluorescein dextran 4 (FD4, Sigma; 4000 Da) was dissolved in egg water toa final concentration of 2 mg/ml. At 2, 3 and 4 dpf, casper embryos weremicroinjected with FD4 into the caudal vein. Leakage of FD4 wasmonitored with confocal microscopy every 10 minutes after injection until120 minutes post-injection, as described previously (Xie et al., 2010).

Immunohistochemical stainAfter the first screen with fluorescence microscopy, the preciselocalization and cellular composition of the clusters was determined.Stored infected embryos were stained with anti-L-plastin (Herbomel et al.,1999; Bennett et al., 2001) or anti-acetylated tubulin (Wilson et al., 1990).Anti-L-plastin stains phagocytic cells, whereas anti-acetylated tubulinstains the axonal tracts and commissures of the CNS of the zebrafish(Wilson et al., 1990). With the anatomical atlas developed by the zebrafishworkgroup of the University of London (Zebrafish Group and UniversityCollege London, 2009), we determined important anatomical features thatwere needed to define the localization of granulomas more precisely(Fig. 2G).

In short, embryos and larvae were rinsed with 1% PBTx, which contains1% Triton X-100 in PBS, permeated in 0.24% trypsin in PBS and blockedfor 3 hours in block buffer, which is 10% normal goat serum (NGS) in 1%PBTx. Incubation with the first antibody was done overnight at roomtemperature (RT) with anti-L-plastin [1:500 (V/V) dilution] or anti-acetylated tubulin [Sigma T7451, 1:250 (V/V) dilution] in antibody buffer,which is PBTx containing 1% (V/V) NGS and 1% (W/V) BSA. Afterwashing again with PBTx and incubation for 1 hour in block buffer,embryos were incubated in the second antibody overnight at 4°C. For L-plastin staining we incubated in Alexa-Fluor-488 (Invitrogen A11034, 1:200dilution) or Alexa-Fluor-633 goat-anti-rabbit antibody (Invitrogen A21070,

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1:200 dilution); for acetylated tubulin staining we incubated in Alexa-Fluor-488 goat-anti-mouse (Invitrogen A11001, 1:200 dilution). Supplementarymaterial Fig. S3 shows the number of embryos and larvae stained with anti-L-plastin or anti-acetylated tubulin.

Confocal microscopyAfter immunohistochemical staining, embryos were embedded in 1% low-melting-point agarose (Boehringer Mannheim, 12841221-01) dissolved inegg water (60 μg/ml instant ocean see salts) in an 8-well microscopy μ-slide(http://www.ibidi.com). Analysis was performed with a confocal laserscanning microscope (confocal: Leica TCS SP2 with AOBS, microscope:Leica DM IRE2). ImageJ software was used to adjust brightness andcontrast and create overlays.

Histopathological analysisHistopathological analysis was performed on sections of 1-year-old adultzebrafish, which were i.p. infected with different M. marinum strains duringexperiments performed previously in our laboratory (Appelmelk et al., 2008;Stoop et al., 2013). In addition, we infected casper embryos at 1 dpf with M.marinum for histopathological analysis of granulomas in the brain area.Adult zebrafish [8 wpi (±6 days)] and embryos (5 dpi) were anesthetizedwith MS222, fixed in 4% paraformaldehyde in PBS and horizontallysectioned. Sections of 5 μm were mounted on glass slides and stained withhematoxylin and eosin (HE) or with Ziehl-Neelsen (ZN).

Graphs and statistical analysisGraphs in Figs 2, 5 and 6 were made using GraphPad Prism 5.0. Fish wereanalyzed for each infection route and bars represent mean and standard errorof the mean (s.e.m.). Statistical analyses were performed with the sameprogram, using a one-way ANOVA followed by a Bonferroni’s multiplecomparison test.

AcknowledgementsWe thank Gunny van den Brink-van Stempvoort, Theo Verboom, Wim Schoutenand Janneke Maaskant for technical assistance. We thank F. G. Wouterlood andE. Timmermans-Huisman (department of Anatomy and Neurosciences, VUUniversity Medical Center) for assistance with the confocal microscopy. The anti-L-plastin antibody was a kind gift of Prof. P. Martin (Bristol University, UK).

Competing interestsThe authors declare no competing financial interests.

Author contributionsL.M.v.L., M.v.d.K., W.B., A.M.v.F. and A.M.v.d.S. conceived and designed theexperiments. L.M.v.L. performed the experiments. S.A.Y. and A.d.B. performed thehistopathological experiments and analysis. L.M.v.L., M.v.d.K., W.B., A.M.v.F. andA.M.v.d.S. wrote the paper.

FundingThis research was partially funded by the ESPID/Wyeth fellowship 2010-2012(awarded to M.v.d.K.).

Supplementary materialSupplementary material available online athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.015453/-/DC1

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