Review article

Molecular findings and approaches spotlightingMycobacterium bovis persistence in cattle

Angel H. ALVAREZ, Ciro ESTRADA-CHAVEZ, Mario Alberto FLORES-VALDEZ*

Centro de Investigacion y Asistencia en Tecnologıa y diseno del Estado de Jalisco (CIATEJ) A.C.,Unidad de Biotecnologıa, Av. Normalistas 800, Col. Colinas de la Normal, Guadalajara, Jalisco,

C.P. 44270, Mexico

(Received 22 September 2008; accepted 11 February 2009)

Abstract – Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (M. bovis) are the etiologicalagents of human and bovine tuberculosis (TB, bTB) respectively, and share genetic identity over 99%at the whole genome level. Progress has been made towards explaining how mycobacteria and theirinfected hosts remain in balance without producing clinical symptoms of disease, a phenomenonreferred to as latency or persistence, which can be mimicked by certain in vitro conditions. Latency/persistence has mainly been studied using Mtb, where the two-component signalling system, dosRS,has been assigned an instrumental role, and even constitutes the current basis for development of newdiagnostic methods and treatment addressing this particular stage of TB.M. bovis conserves homologgenes that in Mtb play a role in human latent TB infection and that, by analogy, would allow it toenter a persistent state in infected cattle; nevertheless, little attention has been paid to this stage inbovine hosts. We suggest that many of the advances acquired through the study of Mtb can andshould be taken into consideration by research groups and veterinary professionals dealing with bTB.The study of the infection in bovines, paying particular attention to defining the molecular andcellular markers of a M. bovis persistent infection in cattle, presents great opportunities for thedevelopment and trial of new diagnostic tests and vaccines, tools that will surely help in promotingeradication of bTB in high-burden settings.

tuberculosis / Mycobacterium / persistence / latency / DosR

Table of contents

1. INTRODUCTION

Tuberculosis is a mycobacterial infection thataffects a wide range of mammals. Human andbovine tuberculosis (TB and bTB respectively)

1. Introduction........................................................................................................................................... 12. Is M. bovis persistence affected by immune responses in cattle? ....................................................... 33. Mycobacterial factors required for latency .......................................................................................... 54. The dosRS regulon is conserved between M. bovis and Mtb.............................................................. 65. Current assays and strategies for improving human latent tuberculosis diagnosis............................. 66. Challenges in defining persistent bovine tuberculosis......................................................................... 87. Concluding remarks and future directions ........................................................................................... 9

* Corresponding author: Floresv@Ciatej.net.mx,floresvz@yahoo.com

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share key aspects such as developing similarlesions and immune responses, which oftenresult in colonization and spread to the sameorgans, namely lungs and lymphatic tissues[12, 14, 73, 109]. Like in humans, aerosolizedbacteria are the most common source of infec-tious organisms in cattle, and the primary siteof natural infection is the respiratory tract [14,21]. In bTB, airborne infection is consideredthe most common route of transmission, andmore than 19% of cattle with bTB shed themycobacteria [24, 111], mainly early duringinfection [68, 70]. The intermittent nature ofbacilli shedding from infected animals, after ashort initial post-infection period has been doc-umented [67, 68]. Several studies indicate thatbetween 8–26% of naturally infected animalsrelease mycobacteria in clinical samples, butthe precise duration of the secretion period isunknown [75, 95, 111].

Mycobacterium bovis (M. bovis) is a closerelative of Mycobacterium tuberculosis (Mtb),and they share genetic identity over 99% atthe whole genome level [34]. Different studieshave supported the fact that although slight dif-ferences are found in the genome sequence ofMtb andM. bovis, the physiology and host rangespectrum is different [36, 88]. In recent years,progress has been made regarding the molecularbasis that might, at least partially, explain howmycobacteria are able to remain in a persistentor latent infection in its human host, and it hasalso been suggested that this particular stage ofdisease exists in cattle, by analogy with humanTB [14, 42, 84, 109]; nevertheless, until nowlittle attention has been paid to this form ofinfection in bovine hosts.

TB produced by M. bovis is an importanthuman zoonosis associated with consumptionof dairy products contaminated with the bacilli,and to labour risk (such as direct contact ordroplet transmission) in farms or slaughter-houses [25, 39, 105, 108]. Close to 35% ofthe Latin American and Caribbean countrieshave reported the occurrence of TB due toM. bovis, and a conservative estimate wouldbe that it causes 2% of the total pulmonaryTB cases and 8% of extrapulmonary TB cases.Moreover, it is also estimated that 60% of thehuman population live in countries where cattle

undergo no control or only limited control forbTB [20], constituting a potential infectioussource. Ideally, all cattle that give positive read-ings to diagnostic tests should be killed, as theymay well represent a source of ongoing infec-tion. However, under financial constraints, forexample in developing countries, programs tocombat bTB require a more accurate targetingof cattle that represent the greatest threat ofspreading infection [85]. We must considerthe inability of current assays to discriminatebetween active and a likely latent infection incattle, which could make the differencebetween sacrificing truly contagious animalsor only suspected ones.

It was recently shown that M. bovisaccounted for 45% of all culture-positive TBcases in children and 6% of adult cases inSan Diego, California, United States, from1994 to 2005 [93], whereas in the whole coun-try, 1.4% of linked cases were identified as M.bovis [45]. On the other hand, surpassing a pre-viously thought host preference [34], Mtbinfected cattle has been reported in countrieswith high incidence of bTB [16, 105]. Definingthe proportion as well as the clinical presenta-tion of infected subjects (active or persistent)in affected countries is no doubt of enormousimportance, as it is determining the same num-bers for infected cattle [22].

It is worth noting that, contrary to TB, whereactive disease often occurs with clinical signsand symptoms (fever, weight loss, coughing,presence of acid-fast bacilli (AFB) in sputa orother clinical samples), during bTB, M. bovis-infected cattle do not present two well-definedclinical (active or latent) phases, nor does theimmune reactivity test used nowadays (tubercu-lin skin test, TST) has the power to discernbetween these two likely states in infectedbovines. Asymptomatic TST+ cattle have beenfound infected with either M. bovis or Mtb aswell [105].

Clinically, latency is defined as the persis-tence of a tuberculous lesion with viable myco-bacteria in a host without symptoms and hasbeen demonstrated because of reactivation(defined as the onset of active disease upon,e.g., an immunosuppressive event) of appar-ently healthy people and/or reactivity towards

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Mtb antigens despite lack of clinical manifesta-tions and absence of AFB in clinical specimens[30, 35, 53, 54]. In contrast, in the context ofbovines, persistence could perhaps be definedas the existence of a positive skin or IFN-c testin cattle in which no lesions can be detected orwhen a culture is positive in the absence oflesions. However, the first approach presentsthe disadvantage of not directly definingwhether reactivity is due to a present or pastinfection, or even because of contact with envi-ronmental mycobacteria. The latter approach issimply time consuming and again does not nec-essarily address which organisms are causingthe infection unless more specific tests (PCR,hybridization, specific immune assays) areconducted.

Although individuals with latent infectiontheoretically do not transmit the disease, incountries where TB does not have a high inci-dence, it is estimated that there is a risk of 2–23% of reactivation of the disease during theirlifetime, thus constituting a significant sourceof transmission upon reactivation [80]. Reacti-vation is thought to be derived from manyevents that lead to immunosuppression, fromexperimental evidence using Mtb infection ina mouse model [8]. Reactivation of latent tuber-culosis produced byM. bovis infection has beendocumented in people with previous exposureto unpasteurised dairy products [54]. In addi-tion, in TB cases associated with human M. bo-vis infection, the HIV/AIDS pandemic maylead to a greater susceptibility of people to overtTB [39], as has been shown with the use ofanti-inflammatory treatment [54]. Envisioninga similar scenario, should persistent/latent bTBin cattle be present, reactivation could be asso-ciated with changes in cell-mediated immunityin tuberculous cattle as well as with viral immu-nosuppression, metabolic unbalance, corticoste-roids and postpartum period [32, 82, 84, 102].Nevertheless, the association of bTB with per-sistent and immunosuppressive viral diseases,such as bovine viral diarrhoea and bovine leu-cosis, has not been described in spite of itsenzootic character in dairy cattle and deservesfurther attention.

Considering the scarce evidence demonstrat-ing that M. bovis produces a latent/persistent

infection in humans, and suggesting it can pro-duce a similar state in cattle, as well as the fewreports intended to decipher and precisely defin-ing the existence of a chronic/persistent M. bo-vis infection state during disease, and its impactfor uncontrolled bTB in several countries, wepresent this work, pursuing the following goals:(1) to present a brief summary of the currentstatus and advances on tuberculosis persistenceboth in bovine and humans, and (2) to suggestpaths to converge such knowledge for the ben-efit of the diagnosis and treatment effortsagainst bTB, for their application in the veteri-nary field.

2. IS M. BOVIS PERSISTENCE AFFECTEDBY IMMUNE RESPONSES IN CATTLE?

In a histopathological setting, bTB spreadsnot only from animals with gross lung lesions,but also from those considered to represent alatent-like (persistent) infection without visible(gross) lesions [65, 69, 75, 84], although cau-tion should be taken considering the argumentsexpressed above, for the need of clearly demon-strating (not suspecting) a latent bTB phase.Post mortem analysis of samples from cattlewith a positive tuberculin skin test (TST+)and IFN-c test reactors often correlates withgross lesions mainly in tracheobronchial, medi-astinal, and retropharyngeal lymph nodes [76].The majority of bTB cases occurring as naturalinfection are restricted to the lower respiratorytract, but no cavitation of lungs has been found,as opposed to some chronic human TB cases[19, 71, 75, 76, 118], raising exciting questionsas to what is the nature of such differences.Exhaustive histopathological analyses of lungshave allowed the identification of lesions in70% of bTB cases without visible lesions inthis organ [65]. However, under low prevalenceconditions, close to 60% of animals with TST+have no visible lesions, and may thus constitutea persistently infected population, providing agood source of material for experimentallyaddressing the existence of such state incattle.

In order to define the existence of a particu-lar infection stage, a proper characterization and

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classification of the lesions is necessary, as isbeing proposed for the different stages of gran-uloma development [76]. These are as follows:stage I/II early non-necrotic lesions; stage IIIinfected core surrounded by multiple concentriclayers of macrophages (including epithelioidand giant cells) and fibrous tissue; and stageIV central caseous necrosis. It is common tofind type IV in combination with type I/IIlesions [57]. Since caseous necrosis and calcifi-cation accompanied by a small number of posi-tive AFB are common features of granulomasencountered in natural infections, this can bedetected in animals with no visible or isolatedhistopathological lesions, independently of theirTST status or IFN-c responses [57, 69, 70].Changes in the TST status, histopathologicalfindings or cellular markers might prove to behelpful in estimating what type of infection abovine has, either active or persistent, and areproblems that surely deserve further explora-tion, as long as they can be improved to specif-ically determine M. bovis presence or any othermycobacteria as agent of the disease.

At a cellular level, once inside the host,M. bovis is ingested by macrophages and theiractivation aids limitation of or destruction ofinfecting mycobacteria [23, 86]. Moreover, dif-ferences in the antibacterial capacity of bovinemacrophages, depending upon microenviron-ment and organ-specific factors or genetic back-ground, may exist [87]. Experimental datasuggests that an excessive immune responseor a high virulence of the bacilli favour thepathogenesis of bTB [116, 117]. In susceptiblehosts, the interaction ofM. bovis with cells fromthe innate and adaptive immune response in therespiratory tissue and lymph nodes results incolonization and infection with developmentof several-stage lesions leading to the produc-tion of granulomas [76]. Granulomas representthe focal expression of the inflammatoryresponse aimed at preventing mycobacterialgrowth, thus constituting a means whereby bac-teria could potentially be directed or primed toenter a persistent state. During this interaction,T-cells are among the first cells involved inearly granuloma formation [13]. All of the mainT-cell subsets (cd T-cells, CD4+ and CD8+ abT-cells) have been shown to be involved in the

anti-mycobacterial immune response in cattle[85]. There, both CD4+ T-cells and CD8+T-cells have an important involvement in con-trolling infected cells [85]. Interestingly,CD8+ cells may play a deleterious role by con-tributing to the immunopathology of bTB[110], while WC1+cd T-cell may contributetoward the Th1 bias of the immune response inbTB, although they do not appear to be essentialfor the development of delayed-type hypersensi-tivity (DTH) [49, 50]. Within granulomas, thereis a balance between mycobacterial killing andsurvival [99] that results in eradication, contain-ment (persistence, latency) or progression ofthe infection, according to which side of theimmune response is favoured [85, 86].

Within granulomas, the presence of AFBorganisms might suggest these bacteria are ina replicative, rather than a dormant or persistentstate, based on the lack of AFB staining in MtbH37Rv under in vitro latent conditions, inducedby nutrient deprivation [3]. However, we mustpoint out that there is a lack of data about theacid-fast staining properties of bacilli assumedto be genetically impaired to enter a latent state(i.e. dosRS knock-out, a two component systeminvolved in Mtb latency, described later),although it was recently demonstrated that theAFB phenotype is dependent on cell wall lipidmodifications, controlled by phoP [115] andkasB [9]. PhoP was reported to affect expres-sion of DosR-controlled genes [37], and forkasB either direct or indirect control by dosRShas not been demonstrated.

It was recently found that depending on thecytokine profile (either predominantly Th1- orTh2-type) cattle experimentally infected withM. bovis present different degrees of histopa-thological damage, thus strengthening the criti-cal role of the immune response in the outcomeof bTB [106]. As a consequence of hormonalchanges, immunosuppression might occur dur-ing peripartum, diminishing cytokines and anti-bodies, along with Th2 polarization of immuneresponse [63], which in persistently infectedcows might promote reactivation. Further stud-ies have indicated the role of additional factorsin the onset and determining susceptibility tobTB, such as early production of IL-4 secretedby Th2 lymphocyte subsets [89, 90], and also

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the early decrease of IL-10 secretion [106]. Inmycobacterial infections, changes in theimmune balance from a dominant Th1-typeresponse towards a more prominent Th2response are associated with disease progres-sion, B-cell antibody production and anergyof cell-mediated immune responses [85].Recent attempts to establish immune profilesassociated with M. bovis infection status havebeen made in cattle suggesting innate immunegene repression in PBMC from bTB infectedanimals [66]. Despite this attempt, it is clear thatimmune phenomena in bTB are highly complexand hard to characterize in cattle. This outlinesthe need of modifying and improving the cur-rent strategies employed to determine bTBinfection status, perhaps moving towards find-ing and validating specific molecular markers.

3. MYCOBACTERIAL FACTORSREQUIRED FOR LATENCY

The molecular mechanisms responsible forthe onset of active or latent TB or reactivationfrom latency to active TB are not yet wellunderstood. We must consider that mycobacte-ria have a complex life cycle and endure anever-changing environment while trying to suc-cessfully establish and maintain an infection oreven while surviving outside their host waitingfor a new infective process to begin. Neverthe-less, a factor that very likely contributes to thecontinued transmission of TB in humans andperhaps bovine hosts is the demonstrated capac-ity of mycobacteria to exist in a dormant, per-sistent or latent state for extended periods oftime, particularly when under stress [80]. Thisoften leads to dramatic changes in gene tran-scription, including those of particular interestin this review, namely genes related to latency,dormancy or persistence within a hostile envi-ronment. These include but are not necessarilylimited to: (1) Hypoxia, (2) Reactive NitrogenSpecies (RNI), (3) Reactive Oxygen Species(RON), (4) the host immune response (NKcells, activation of TLR and synthesis of hostdefence peptides among others). All these fac-tors impose a combination of stresses that affectthe pathogen [94, 101]. A function that would

be expected to be up regulated during suchattacks is the antioxidant response. In thisregard, genes putatively involved in defenceagainst oxidative stress can be found in myco-bacterial genomes, such as noxR1, noxR3, andahpC, which possess high identity with genesencoding for antioxidant enzymes widely dis-tributed in prokaryotes. The glbN gene, whichcodes for a truncated haemoglobin (trHbN), isresponsible for the nitric oxide (NO) metaboliz-ing activity observed in mycobacterial station-ary phase in vitro cultures [74]. Additionally,other components are required for adaptationsoccurring during persistent infection in animalor cellular models, where the protein a-crystal-lin (HspX, Rv2031c or Acr) from Mtb isrequired during the phase of steady growthwithin macrophages [104, 122].

Studies in both Mtb and M. bovis var. BCGgrown under stress with low oxygen tensionhave shown that HspX is a protein expressedduring these conditions, and is virtually the firstprotein required for a latency-like state func-tionally demonstrated to have an impact in bothorganisms under these particular conditions [33,58, 103].

Different studies have approached the ques-tion of latency using alternative techniques. Forexample, Rustad et al. [97] suggest an extendedhypoxic response regulon, and their studyrequires in vivo validation of the participationfrom genes found in their work. Saxena et al.[100] used a genetic screen for in vivo expressedtechnology; however, it is hard to directly extrap-olate studies where not only transcription ofhypoxia-responsive genes is assessed, but alsotranslational effects (such as altered protein sta-bility ofGFP and/or kanamycin) likely occurringon the reporter genes should be considered.

Furthermore, the roles of MprAB [77], aswell as PhoP [37] recently added another levelof complexity solely to DosR regulation, andare not developed in this work in the idea ofsuggesting markers to define whether or notlatency occurs in cattle, conversely to definingthe mechanisms regulating an event that it isstill unclear in bovines. Considering these argu-ments as well as the evidences linking in vitroto in vivo relevance for DosR and DosR-regu-lated genes, including the potential use of some

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DosR-regulon proteins as new markers forlatent/persistent infection, we discuss its partic-ipation in further detail below.

4. THE dosRS REGULON IS CONSERVEDBETWEEN M. BOVIS AND MTB

The dosRS (also known as devRS) two-com-ponent system of Mtb regulates the expressionof a set of 48 genes induced either by hypoxicshift-down or exposure to low doses of NOin vitro [114] and constitutes an operon withcomplex regulation that participates in bacterialresponse against a wide range of stresses [92].DosR is the response regulator with DNA-bind-ing activity, and DosS is one out of two SensorHistidine Kinases (HK), along with DosT,which phosphorylates and thus regulates DosRDNA-binding activity. Its relevance in myco-bacteria was independently confirmed by twogroups, using M. bovis var. BCG (a M. bovisstrain where a set of genomic deletions, inser-tions, and duplications have occurred as a con-sequence of in vitro passages) as a modelorganism [10], and then confirmed in Mtb[79], where mutants lacking either one or bothcomponents are affected in their capacity to sur-vive a long-term culture under oxygen-limitingconditions. On the other hand, conflictingreports about its participation during infectionin animal models have been published [62,78], perhaps attributable to the different modelsused by each group. Indeed, Converse et al.[18] analyzed the participation of the dosRSsystem in four different models ofMtb infection(rabbits, guinea pigs, mouse hollow-fibre, andmouse), and found a growth defect only inmouse and guinea pigs. Kesavan et al. [51] ana-lyzed expression of DosR-regulon and non-DosR controlled genes during Mtb persistentinfection in rabbits. Another analysis of myco-bacterial response to hypoxia and exposure tonitric oxide, including 14 different vaccinestrains of M. bovis var. BCG, revealed the pres-ence of a functional DosR-regulon similar tothat found in Mtb [59]. Nevertheless, severalBCG vaccine strains lacked narK2 and narXinduction and exhibit an altered phenotypesduring dormancy [46]. We should notice the

lack of immune responses to DosR regulon pro-teins following M. bovis var. BCG subcutane-ous vaccination in a mouse model, and evenin humans [35, 59], whereby such lack ofinduction might be the consequence, at leastpartially, of reduced bacterial survival with aninadequate space-temporal antigen productionand presentation.

The genome ofM. bovis strain AF2122/97 isvirtually identical (99.95%) to that of Mtbstrains H37Rv or CDC1551 [34]. By in silicoanalysis, comparing the predicted proteinsequences derived from the AF2122/97 andH37Rv DosR antigens, we found an average97% identity at the amino acid level in 41 outof 48 proteins, with only one amino acid differ-ence in the reported sequences of DosS, withina predicted GAF domain. GAF presents haem-binding activity [98]. The other HK, DosT,already shown to phosphorylate DosR in vitro[91] and also relevant for survival under hyp-oxic conditions [52], also has one amino aciddifference within a GAF domain, whereasDosR is 100% identical. This constitutes astrong indicator of the potential capacity ofM. bovis to induce a bacterial survival mecha-nism that would enable it to remain present incattle in a latent state. We can question whetheror not differences in such GAF domains, oreven in the rest of the proteins belonging tothe DosR-regulon, have a biological role inmycobacterial adaptation to a different host.

5. CURRENT ASSAYS AND STRATEGIESFOR IMPROVING HUMAN LATENTTUBERCULOSIS DIAGNOSIS

For many decades, the TST assay, using apurified protein derivative (PPD), has been themost commonly used test to check for tubercu-losis in cases of subclinical infection, both inhumans and in cattle, with a little variation inthe latter by comparison of reactivity towardPPD from M. bovis versus PPD fromMycobac-terium avium. Mycobacterial PPD, as reportedelsewhere, consists of a mixture of several hun-dred secreted proteins, obtained by trichloroace-tic acid precipitation of cell-free culture filtratesderived from pathogenic mycobacteria. Due to

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inherent genomic variations, it is tempting tospeculate over differences in PPD constitutionfor Mtb, M. bovis and M. avium, for example,affecting specificity of detection, although adetailed analysis (in silico or proteomic) thatensures such a prediction should be conducted.

The use of PPD has a major drawback, asmost antigens present in it are also present inM. bovis var. BCG, a bacterial strain used to vac-cinate humans at an early age, which may affectthe specificity of the test [6]. Despite this, TST isstill considered the best tool for classifying indi-viduals with no symptoms of clinical TB aslatently infected [64]. Paradoxically, the proteinsused as antigens in TST are produced by myco-bacteria under conditions not experimentallydefined as persistent. In spite of this fact, theiruse is considered in some developed countriesas a valuable means of diagnosing both clinical(active) and subclinical (latent) TB [2, 6, 15]. Itwas not until very recently that new tests startedto emerge for determining TB infection status[6, 17]. These are based on the detection ofIFN-c in blood samples after in vitro stimulationof blood cells with the so-called specific Mtbantigens ESAT-6 and CFP-10 [5], which areconsidered specific due to their absence in M.bovis var. BCG used in human vaccinations.This assay is the basis of a kit commerciallyavailable for determining human infection(QuantiFERON�-TB Gold; Cellestis Limited,Carnegie, Victoria, Australia). In humans, astudy carried out in India based on measuringthe cellular response (IFN-c production) trig-gered by ESAT-6 and CFP-10 of Mtb, indicateda prevalence of 80% of latent TB infection in anapparently healthy population used in the study[53]. However, ESAT-6 and CFP-10 are con-served in wild-type M. bovis, and do not allowthe determination of which pathogenic myco-bacteria (Mtb or M. bovis) is infecting humansunless time-consuming procedures of microbio-logical culture, biochemical and antibiotic sus-ceptibility tests or PCR approaches, not yetstandard in some settings, have been conducted.In this regard, patients who had a negativemicrobiological cultures and normal lung his-tology were found to contain the insertionsequence (IS) 6110, a characteristic region ofthe tubercle bacilli, by in situ PCR [43].

On the other hand, proteins belonging to theDosR-regulon, which are expressed underexperimental conditions considered to mimicaspects of latency, have also been evaluated astargets that differentiate TB infection status[30, 35]. HspX proved to be a good stimulatorof the cellular immune response in patients withasymptomatic tuberculosis [30, 35] thus promis-ing to be a new, good, and specific marker forlatent TB infection that could replace the TST.However, this protein was immunogenic for Bcells of patients with active pulmonary tubercu-losis as well [119], and it is still unknownwhether or not HspX could indeed be a goodmarker of latent TB infection at this point.Therefore, three proteins of the DosR-regulon,namely Rv1733c, Rv2031c (HspX), andRv2626c, were evaluated in an independentstudy, and it was found that they induced a goodcellular immune response in a mouse model oflatency [96]. Furthermore, in asymptomatic,TST+ individuals from the Netherlands, a coun-try with low incidence of TB (TST+, being amarker of latent infection), the immune responsefor four DosR-regulon proteins (Rv1733c,Rv2029c, Rv2627c and Rv2628c) was highercompared to that of cells from healthy peopleand patients with active disease [56].

In a different context, a role for cell typesdistinct to macrophages possibly linked to TBlatency has started to emerge, based on in situPCR evidence showing Mtb DNA in type IIpneumocytes [43] or human adipose tissue[72]. Together, all this evidence expands alter-natives for monitoring the infection status ofsubjects suspected to contain latent mycobacte-ria. Likewise, when bacilli are inside type IIpneumocytes, the heparin binding haemaggluti-nin (HbhA, not part of the DosR-regulon), wasfound to be strongly expressed [28, 81]. Astudy measuring IFN-c production in responseto stimulation with native HbhA in 205 personsin Belgium (a country with a low incidence ofTB where vaccination with BCG is not rou-tinely used) gave a 92% sensitivity and a spec-ificity of 94% using HbhA, compared to 90%and 70% obtained with PPD for detection ofinfected subjects, regardless of their BCG vac-cination status as opposed to PPD reactivity.This indicates that in this setting, HbhA is a

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good antigen for detecting individuals withasymptomatic infection [48]. As methylationof native HbhA is important for T-cell stimula-tion, a recombinant protein for wide use shouldpossess this modification.

Thus, published evidences suggest thatHbhA and DosR-controlled proteins might begood candidates for discriminating betweenactive TB and latent infection. In fact, someDosR-regulated antigens have been either sug-gested or already used as new sub-unitary orDNA vaccines [7, 60, 96]. Perhaps it is abouttime to redefine the gold standard for TB infec-tion status.

6. CHALLENGES IN DEFININGPERSISTENT BOVINE TUBERCULOSIS

In humans, active disease is clearly shownby clinical findings such as weight loss, nightfevers, and coughing. Conversely, latent infec-tion is demonstrated because of the reactivityto selected mycobacterial antigens with no signsof disease [30, 35, 53]. In cattle, there is noclear sign of a different status of active versuslatent infection, and the concept of latent infec-tion has remained more as a speculation than aproven fact [14, 42, 84, 109]. Perhaps this isinfluenced by the relatively short life span ofcows compared to humans, or the lack of ethi-cal concerns about sacrificing infected individu-als, with greater concerns about economicallosses and no attention to better understandingthe infective process. However, this latter pointdoes not consider continued transmission toanimal and human hosts.

Latency has been suspected in animals that,upon exposure, turn positive to TST or anIFN-c release assay using bovine PPD,purified ESAT-6/CFP-10 or the commerciallyavailable kit based on IFN-c (BOVIGAM,Prionics, Zurich, Switzerland) [1, 120] that alsouses PPD [14]. This complicates theinterpretation and diagnosis of animals trulyinfected with human pathogens or environmen-tal mycobacteria [47, 107]. In fact, PPDinduced cross-reactions that varied accordingto the area, ranging from 2 to 20% [40, 41].Evidence suggesting that diagnostic assays

(TST and IFN-c tests) can detect bTB incattle soon after M. bovis infection regardlessof the dose has been published [27, 70] thus,none of the tests currently available for thediagnosis of bTB allow a perfectly accuratedetermination of the M. bovis infection statusof cattle [26]. Other authors indicate that duringcow-to-cow M. bovis transmission, it takesabout two months to present a TST+ reaction(ranging from 8 to 65 days) [38], potentiallyincreasing the number of false negative results.Moreover, reversion from a TST+ result occursat a 5% rate per year [38], possibly indicatingeradication of infection or simply waning ofimmune memory.

As an alternative, an enhanced version ofTST, which includes determination ofMPB83-specific antibody responses, was devel-oped and found to have a positive correlationwith bacterial loads in infected cattle [61].Denis et al. [31] used a different strategy byincluding immune modulators in an IFN-crelease assay, and found that anti-IL10 antibod-ies increased sensitivity of detection. However,these improvements are far from establishingwhether or not latency occurs during bovineinfection, thus further strengthening the needof either using antigens that allow this distinc-tion for human TB and see how do they workin the veterinary setting, or finding new anti-gens that help to determine the status (activeor persistent) of infection in cattle. We mustalso consider that animals naturally show vari-ability in TST responses, and some show noclinical signs of disease or visible lesions whenslaughtered, with microbiological culturesbeing negative or positive but from lymphnodes only. Some cows have the ability to resistovert infection despite exposure to M. bovis.The significant numbers of TST+ cattle thathave no evidence of infection at abattoir meatinspections or on laboratory examinationswould support, at least in part, the idea of alatent infection during bovine tuberculosis [84].

Although BCG has been shown to be aneffective vaccine against bTB even using alow dose in cattle [11], it cannot be widely usedunless a cost-effective laboratory test discrimi-nates between infected and vaccinated cattle.IFN-c responses to PPD are induced by BCG

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vaccination of cattle, but their magnitude andduration are dose-dependent and short-lived[4]. Some antigens are either not present (i.e.ESAT-6 or CFP-10) or are only weaklyexpressed (i.e. MPB64 or MPB70) in BCG,and their use in the IFN-c assay has beenshown to offer an opportunity to discriminatebetween animals infected with M. bovis andthose vaccinated with BCG [44, 83, 112]. Quiteinterestingly, T cell-responses against ESAT-6correlated better with disease severity in cattle[113].

In addition to the lack of clinical symptoms,it is known that livestock is likely to present aTST+ reaction because of exposure to non-pathogenic, environmental mycobacteria [47,107], thus increasing the rates of false positivedetection. Recently, evidence of the effect ofenvironmental strains of M. avium interferingwith immune responses blocking BCG multipli-cation and induction of protective immunity totuberculosis has been found [29, 121].

Further complications in defining which ani-mals are indeed infected (actively or persis-tently) arise from the phenomenon of anergy.The origins of anergy in tuberculous individualshave not been completely defined but it isattributed to immunosuppression [55] and morerecently to an undefined type of tolerance [47,121]. In particular, in cattle under intensive pro-duction, the cases of anergy could be related tostressing factors such as transportation, han-dling, metabolic unbalance, and treatments withprostaglandins or corticosteroids, among others[32]. A post-mortem study with healthy, TST+animals, with no visible injuries, indicated thatthe expression of interleukin IL-4 d3 in theseanimals may be playing an important role inmaintaining protection against infection afterexposure to M. bovis [89, 90]. Therefore, thiscellular response could be considered a particu-lar hallmark of latent infection in cattle, consti-tuting a plausible biomarker from the host’spoint of view.

Clearly the existence of animals with asymp-tomatic bTB imposes a great disadvantage tothe veterinary field, further supporting the needfor well established testing markers to distin-guish active versus latent infection in cattle.These tests need to either directly measuring

gene expression from M. bovis isolated fromwell-defined histopathological sections, or eval-uating cellular markers from infected animals inlow and high-incidence settings of bTB orexperimentally infected in a time-course man-ner as already started by Thacker et al. [106].Similar to latent tuberculosis in humans, in cat-tle, latency represents a significant challenge forthe eradication of bTB, through the possiblereactivation in an infected animal [14]. Despitethe current progress achieved in the study oflatent tuberculosis in humans, defining latencyin bTB has practically and inexplicably beenignored, except for a recent call to define theparticipation of latency in the control of bTB[42]. It is tempting to envision that cattle maybe the species in which persistent infection bet-ter resembles human latency. However, meth-ods for experimental induction of persistenttuberculosis in cattle do not exist yet [109].

7. CONCLUDING REMARKSAND FUTURE DIRECTIONS

Latent tuberculosis infection remains a pub-lic health problem, because reactivation canhappen many years after primary infectionresulting in renovated transmission of strainspreviously thought to have been gotten rid of,or that existed thoroughly unsuspected. Despitelatent TB being recognized in humans for along time, persistent infection in cattle has lar-gely been ignored. A comparative genomicspoint of view could be quintessential in provid-ing better means to control, diminish or eradi-cate epidemiologically relevant events. Itwould be very interesting to investigate whethermonitoring expression of genes belonging tothe DosR-regulon in M. bovis-infected cattlewould finally allow a description of a latent/per-sistent phase of bTB. Using this approach incombination with analysis of the expressionand role of mycobacterial genes required foractive growth would allow a more precise def-inition of temporal profiles during transitionstages of the infection. One of the most activelines of basic research in the field of tuberculo-sis consists of deciphering what genes areturned on or off when mycobacteria move from

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an active and replicative state to one ofpersistence.

Defining whether latency occurs in cattleand assessing the potential participation of dif-ferent cell types as ‘‘incubators’’ of persistentbacilli can lay the groundwork for identifyinggenes and/or proteins that may be useful bothfor diagnosis and for the development of newvaccines against human and bovine tuberculo-sis. Alternatively, infected organs, from wellcharacterized stages of infection in cattle arehighly abundant in endemic areas and constitutean excellent source for which gene-specific orgenome-wide expression profiles using ultra-sensitive real time reverse transcription andquantitative PCR can be used to monitor theexpression of genes required for active or latentinfection based onMtb studies, allowing dissec-tion of their participation in a natural setting.

Due to their relevance for humans, studies oflatency/persistence or isolated factors linked toit (i.e. hypoxia) have solely been studied intwo slow-growing mycobacteria (Mtb andM. bovis var. BCG), but it might well be thatother organisms within this genus are able tosustain such an infection, either in humans ortheir natural/preferred hosts. It would be inter-esting to look at different organs in chronicallyinfected organisms, and perhaps conduct a spe-cies-wide analysis of the microbes found there,using markers such as ribosomal genes forexample, or those that better and more accu-rately show the population represented in thoseenvironments.

On the other hand, addressing immune pro-files or host susceptibility factors, either inexperimentally- or naturally-infected cattle,would be a way to start unravelling the partici-pation and balance between bacteria and bovinehost immune system, whereby a persistent stateand its contribution to continued transmissionduring long periods of time despite lack of evi-dence of ill animals could be ascertained. Thus,detailed histopathological or even molecularanalyses are required to discern how manybTB lesions harbour mycobacteria in an activeand/or persistent/latent state. Further experi-ments linking immunological profiles to thepresence of mycobacteria in the different typesof lesions, with demonstration of bacterial

expression of latency markers are required.An immune response profile assessed in cowswith diverse degrees of pathology, which corre-lates with contained infections of minimal tis-sue damage, might indicate which host factorsare important for maintaining a persistent,non-progressive infection.

Considering the rapid accumulation andavailability of genome sequences, we coulduse this information to answer whether thereis any biological meaning to possible changesin DosRS sequences of vaccine and mycobacte-rial strains, and their likely impact on efficacyof protection and virulence, respectively. Giventhe variety of insertion, deletion and duplicationevents that occurred in BCG strains, the differ-ences found in DosS and DosT, key compo-nents of a system developed to sense andrespond to oxygen limitations or immuneresponse-derived stressors, might have animpact in how mycobacteria adapts to its host.Mapping of DosRS and DosT sequences in dif-ferent environmental pathogenic strains of M.bovis, for example, should be undertaken toassess genetic variability in these genes. Moni-toring expression of these genes in the sameisolates and/or infected tissues would reveal sig-nificant information about latency in cattle. Wemust consider that it is very likely that genesrequired for dormancy in Mtb which have avery close homolog in M. bovis, might beexpressed in vivo during bTB.

The participation of genes other than theDosR-regulated ones cannot be ruled out, buttheir participation and relevance need to be fur-ther studied and validated in infected subjects.Studies showing the presence of humanslatently infected withM. bovis indicate this bac-terium may be able to persist in such a form in anon-preferred host, thus strengthening thenotion of a possible persistent form in its naturalhost, the bovine, where it may also be latentlyharboured. Furthermore, Mtb has been isolatedfrom asymptomatic cattle, suggesting the samecapability of this bacterium to cause a silentinfection in these animals. On the other hand,as no clinical symptoms are developed ininfected bovines, this imposes a harder chal-lenge for trying to establish evidence of latent/persistent infection in cows. The occasional

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histological findings in slaughterhouses, joinedto TST assays in cows, constitutes by no meansa way to determine if M. bovis lies dormant incattle and what the impact of such a state mayhave on the continued transmission of bTB.This clearly indicates that we need to focusour attention on its close relative and use theknowledge gathered there to speed up advancesfor the veterinary field dealing with tuberculosisevery day.

Acknowledgements. We would like to thank Dr HugoBarrios for his helpful comments and review ofthe manuscript; we also thank Mrs Dana ElizabethErickson and Mr Winston Smith for their critical read-ing and comments to improve this work.

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