research article evaluation of cocktails with...

Research ArticleEvaluation of Cocktails withRecombinant Proteins of Mycobacterium bovis fora Specific Diagnosis of Bovine Tuberculosis

Mariacutea Laura Mon1 Roberto Damiaacuten Moyano1 Mariana Noelia Viale1

Mariacutea Alejandra Colombatti Olivieri1 Ignacio Joseacute Gamietea2

Valeria Noely Montenegro1 Bernardo Alonso3 Mariacutea de la Paz Santangelo1

Mahavir Singh4 Rosario Duran5 and Mariacutea Isabel Romano1

1 Instituto de Biotecnologıa INTA Los Reseros y Nicolas Repetto 1686 Hurlingham Buenos Aires Argentina2 INTA Estacion Experimental Agropecuaria Delta 1670 Delta Buenos Aires Argentina3 SENASA Direccion de Laboratorio y Control Tecnico (DILAB) 1640 Martınez Buenos Aires Argentina4 LIONEX Diagnostics ampTherapeutics GmbH 38126 Braunschweig Germany5Unidad de Bioquımica y Proteomica Analıtica Institut Pasteur de MontevideoInstituto de Investigaciones BiologicasClemente Estable 11400 Montevideo Uruguay

Correspondence should be addressed to Marıa Isabel Romano romanomariaisabelintagobar

Received 7 March 2014 Revised 21 May 2014 Accepted 6 June 2014 Published 8 July 2014

Academic Editor Armando Acosta

Copyright copy 2014 Marıa Laura Mon et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis(TBB) The specificity of these diagnoses however is compromised because both are based on the response against purifiedprotein derivative of Mycobacterium bovis (PPD-B) In this study we assessed the potential of two cocktails containing M bovisrecombinant proteins cocktail 1 (C1) ESAT-6 CFP-10 andMPB83 and cocktail 2 (C2) ESAT-6 CFP-10MPB83 HspX TB103 andMPB70 C1 C2 and PPD-B showed similar response by DTH inM bovis-sensitized guinea pigs Importantly C1 induced a lowerresponse than PPD-B inM avium-sensitized guinea pigs In cattle C1 displayed better performance than PPD-B andC2 indeed C1showed the least detection of animals either vaccinated or Map-infected To optimize the composition of the cocktails we obtainedprotein fractions from PPD-B and tested their immunogenicity in experimentally M bovis-infected cattle In one highly reactivefraction seven proteins were identified The inclusion of FixB in C1 enhanced the recognition of M bovis-infected cattle withoutcompromising specificity Our data provide a promising basis for the future development of a cocktail for TBB detection withoutinterference by the presence of sensitized or infected animals with other mycobacteria

1 Introduction

The infection and disease produced byMycobacterium boviswhich is the causative agent of bovine tuberculosis (TBB)is an important problem in cattle and other animal speciesHuman tuberculosis (TB) is produced mainly by Mycobac-terium tuberculosis HoweverM bovis can also be responsiblefor the disease in humans which makes this bacteriuman important zoonotic species [1] The TBB eradication

programs in Argentina are based on a prompt detection ofinfected animals and their subsequent removal from the herd

Delayed type hypersensitivity skin test (DTH) which isbased on response against single intradermal inoculation ofpurified protein derivative ofM bovis (PPD-B) remains theprimary surveillance tool to diagnose TBB in our countryThe intensity of DTH reactions elicited inM bovis-sensitizedguinea pigs is the model to select batches of PPD-B to beused in the field Thus this animal model could be used to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 140829 12 pageshttpdxdoiorg1011552014140829

2 BioMed Research International

evaluate reagents with potential for TBB diagnosis Diseasecontrol can also be facilitated by using the interferon-gamma(IFN-120574) assay In their original form these tests DTH andIFN-120574 are based on the response against PPD-BThis reagentis a mix of proteins lipids and carbohydrates obtainedfrom heat-killed cultures of M bovis strain AN5 whichcompromise specificity because some of its antigenic com-ponents are present in others nonpathogenic and pathogenicmycobacteria [2 3] Animals infected with Mycobacteriumavium subsp paratuberculosis (Map) the etiologic agentof paratuberculosis (PTB) or vaccinated with BCG havedemonstrated positive response in these assays using PPD-B The specificity of these tests can be increased with the useof definedM bovis antigens

Many mycobacterial proteins have been isolated andeval-uated in DTH and IFN-120574 assays in animals with TBB[4ndash13] For instance ESAT-6 (Rv3875 or EsxA) is one of themajor antigenic targets identified in both cattle and humanswith tuberculosis [14] Several other antigens with molecularmasses of around 10 kDa and belonging to the Esx family ofproteins have been identified subsequently such as CFP10(RV3874 or EsxB) and TB103 (Rv3019c or EsxR) and theyhave been shown to be equally well recognized by T cells[15] ESAT-6 and CFP-10 or combinations of these antigenshave been extensively tested by DTH and IFN-120574 assays innaturally M bovis-infected cattle with satisfactory results[16ndash19] The studies performed in M bovis-infected cattleyielded promising results with cocktails containing ESAT-6CFP-10 plus the addition of other antigens [18ndash20] ESAT-6 and CFP-10 are encoded in a genomic region RD1 thatis absent in BCG Pasteur and thus are useful as diagnostictools for differentiating TB-infected from BCG-vaccinatedanimals (DIVA) [19] Proliferative immune response withsynthetic peptides derived from the sequences of CFP-10and ESAT-6 showed that BCG-vaccinated cattle did notrespond to this peptide cocktail whilst they all responded toPPD-B [21]

Furthermore the use of a recombinant ESAT-6 CFP-10fusion protein was useful to differentiate M bovis-infectedfrom those Map-infected cattle [22] Several studies haveidentified immunogenic proteins present in bothM bovisMtuberculosis [4 9 10 15 17] A disadvantage of those studieshowever is that they are restricted to a relatively small setof proteins biased by the initial selection criteria Recentlya nonbiased approach with an extensive library of clonesexpressing mycobacterial proteins was used and 33 geneproducts that induced IFN-120574 production in whole-bloodcultures from TB-reactor animals were detected One ofthese proteins did not induce responses in BCG-vaccinatedanimals which is suitable for DIVA diagnostic Greaterresponder frequencies were observed for BCG-vaccinatedcattle than for TB reactors with the remaining antigenswhich leads to speculate that other ldquoless immunodominantrdquoantigens maybe recognized to a greater extent in BCG-vaccinated animals [23]

These recent studies show that the search of otherantigens for a more sensitive and specific diagnosis of TBis still relevant Unfortunately few studies have shown thefeasibility ofM bovis antigens to increase specificity of DTH

using either the guinea pigmodel or experimentallyM bovis-infected cattle [24ndash26] Moreover even more informationof the use of cocktails with M bovis antigens in naturallyinfected cattle remains scarce

The present study aimed to assess cocktails containingpreviously defined antigens of M bovis in order to differ-entiate animals with TBB from vaccinated- or Map-infectedanimals For this purposewe used different approachesDTHin the guinea pig model and both DTH and IFN-120574 assays inthe cattle model The specificity of the defined antigens wasevaluated in both BCG-vaccinated and Map-infected cattleFinally novel cocktails enriched with immunogenic proteinsidentified bymass spectrometry analysis of selected antigenicfractions of PPD-B were evaluated in TBB PTB and freeherds

2 Material and Methods

21 Antigens In the first part of the present study we assessedthe application of antigens that have been extensively testedin TBB with satisfactory results These antigens were usedto produce two cocktails cocktail 1 (C1) containing ESAT-6 CFP-10 and MPB83 and cocktail 2 (C2) with ESAT-6CFP-10 MPB83 MPB70 TB103 and HspX The potency ofthe cocktails was evaluated by DTH in M bovis M aviumsensitized-guinea pigs and nonsensitized guinea pigs as wellas in experimentallyM bovis-infected cattle (DTH and INF-120574) BCG-vaccinated naturally Map-infected cattle and in afree TBB and Map herd by IFN-120574

The purified recombinant proteins ESAT-6 CFP-10MPB70 MPB83 TB103 and HspX were kindly provided byProfessor Mahavir Singh (Lionex Diagnostics andTherapeu-ticsGmbHGermany) In all cases the endotoxin content waslt127 IUmg

In the second part we characterized immunodominantantigens from the PPD-B The coding sequences ofCFP2 FixB and PepA were amplified by PCR using thefollowing primers CFP2-fw 51015840-cgcggatccatgaagatggtgaaatcga-31015840(BamHI site) and CFP2-rev 51015840- ataagctttcagttccctgcggcctgc-31015840(HindIII site) FixB-fw 51015840-cgggatccatggctgaagtactgg-31015840(BamHI site) and FixB-rev 51015840-taagcttctagcccttgcgggcc-31015840 (HindIII site) PepA-fw 51015840-aaggatccatgagcaattcgcgccg-31015840(BamHI site) and PepA-rev 51015840-ataagctttcaggccgggggtccct-31015840(HindIII site) M bovis DNA was employed as templatefor the amplifications The amplification programme wasas follows an initial step of 95∘C for 10min 35 cycles of95∘C for 1min 58∘C for 1min and 72∘C for 1min followedby a final termination step of 72∘C for 8min The completeopen reading frames of CFP2 (507 pb) FixB (957 pb) andPepA (1068) were subsequently cloned into the expressionvector pRSET-A (Invitrogen BV Leek The Netherlands)The derived constructions were transformed into Escherichiacoli BL21 (pLys) competent cells A 3-mL inoculum of E coliBL21 harboring pRSET-A with the gene inserts was diluted120 and grown to the mid-logarithmic growth phase(OD of 06) in LB-ampicillin for 16 h at 37∘C One mMconcentration of isopropyl-D-thiogalactopyranoside (IPTG)(Merck Germany) was added to induce recombinant gene

BioMed Research International 3

expression over 3 h Cells were then harvested by centrif-ugation and frozen at minus20∘C until further use The cell pelletwas lysed with 1mL of lysis buffer (Tris-HCl 100mM pH75 NaCl 300mM Glycerol 20 NP40 1) using a FastPrep FP120 40 seg 55ms (Bio101-Savant Holbrook NY)The expression of the soluble antigens was confirmed byboth Coomassie blue staining of polyacrylamide gels andWestern blot analysis using Mouse anti-His antibody asprimary antibody (GE Healthcare UK) and anti-MouseAlkaline phosphatase as secondary antibody (SigmaMissouri USA) The recombinant proteins in the E colicell extracts were purified by using a Ni-NTA resin (Niquelnitrilotriacetic acid) (Qiagen Corp CA) For this purposethe supernatant was incubated for 90 minutes at 4∘C withthe resin and immobilized in the column The resin wasthen washed with lysis buffer and eluted with increasingconcentrations of imidazole (250mM 500mM and 1M)(Sigma St Louis MO) in buffer (Tris-HCl 50mM pH 75NaCl 500mM Glycerol 20 and NP40 1) The presenceand purification of the protein was confirmed by Coomasieblue and Western blot The protein concentration wasestimated with the Micro BSA Protein Assay kit (PierceUSA)

PPD-B and PPD-A were obtained from the National Ser-vice of Agricultural and Food Health and Quality (SENASABuenos Aires Argentina) with a protein concentration of1mgmL and 05mgmL respectively

22 Gene Nomenclature and Identity of Antigens of C1 and C2The cfp-10 gene of M bovis (Mb3904) has 100 identity tocfp-10 of M tuberculosis (Rv3874) which encodes a 10 kDaculture filtrate antigen CFP-10 or EsxB

The esat-6 gene of M bovis (Mb3905) has 100 identityto esat-6 ofM tuberculosis (Rv3875) which encodes an earlysecreted antigenic target 6 kDa ESAT-6 or EsxA

The genes cfp-10 and esat-6 are absent in the genome ofM bovis BCG and Map

MPB83 and MPB70 are highly homologous proteinswithin M tuberculosis complex members and the orthol-ogous in different members of the complex are virtuallyidentical However they are major antigens that are highlyexpressed by M bovis and considerably less abundantlyexpressed by M tuberculosis The mpb83 gene (Mb2898)and mpb70 gene of M bovis (Mb2900) have 100 identitywith their orthologous genes in M tuberculosis (Rv2873 andRv2875 resp)

The genesmpb83mpb70 and hspX inM bovis have 100identity with these genes inM bovis BCG and they are absentfrom Maprsquos genome The hspX gene of M bovis (Mb2057c)has 100 identity with the gene (Rv2031c) that encodes the16-kDa alpha crystalline (Acr) protein of M tuberculosis orheat shock protein X

The TB103 or esxR gene ofM bovis (Mb3045c) has 100identity with the gene (Rv3019c) of M tuberculosis whichencodes a secreted esat-6 like protein

The protein TB103 ofM bovis has 100 identity with thisprotein inM bovis BCG and 80 with the protein of Map

23 Animal Models

231 DTH Skin Test in M avium and M bovis-SensitizedGuinea Pigs Two groups of guinea pigs were sensitized onewith M bovis another with M avium by intramuscularinoculation of 05mL of a sterile heat-killed suspension ofMbovis strain AN5 or heat-killed suspension ofM avium strainD4ER respectively A group of nonsensitized guinea pigs wasused to evaluate the specificity of the reagents The sensitizedanimals were prepared by clipping the hair from the entireabdominal and flank areas Thirty days after injection theguinea pigswere used for testing PPD-B PPD-AC1 C2 Esat-6CFP-10 and buffer phosphate saline (PBS) as a negativecontrol On each guinea pig six sites for injection of PPD-B and the cocktails were selected Three sites on each side ofthemidline and spaced at a sufficient distance (2-3 cm aprox)from each other to avoid overlapping of skin reactions Theinduration diameter in guinea pig was read at 24 h accordingto protocol used by the National Service of Agriculturaland Food Health and Quality (SENASA) Buenos AiresArgentina

C1 and C2 cocktails were prepared in PBS at a final con-centration of 1 120583gmL of each antigen The animals receivedintradermal injections with 02mL of each cocktail or PPD-Band PPD-A at 1 120583gmL

232 Cattle

(1) BCG-Vaccinated and ExperimentallyM bovis-InfectedCat-tle One group of five castrated male Holstein-Fresian calvesof three to four months of age was inoculated subcutaneouslyin the side of the neck with 1 times 106 colony forming units(CFU) of BCG Pasteur suspended in PBS Another group ofsix animals was infected with a wild boar virulent strain Mbovis 04-303 by intratracheal instillation of 5 times 107 CFU asdescribed previously [27] The strain M bovis 04-303 is anisolate obtained from a wild boar with tuberculous lesions[28] Briefly this inoculation procedure was carried out byanaesthetizing the calves with xylazine HCl (Rompun BayerGermany 01mgkg) intravenously and then inserting an80 cm endotracheal tube into the trachea A cannula wasinserted through the endotracheal tubeThe 15mL inoculumcontaining the M bovis 04-303 was injected through thecannula and flushed out with sterile saline equal to 10mLvolume

All the animals used in this study were DTH negative toboth PPD-A and PPD-B at the beginning of the experimentsThese experimentally infected animals were part of a trial thatincluded evaluation of candidate TBB vaccines [27]

To confirm that the animals were successfully infectedthe calves were euthanized 100 days after infection andthen thin slices of lungs and lymph nodes of the head andpulmonary region were analyzed in search of granulomaformations All M bovis-infected animals presented macro-scopic lesions and were positive for both bacterial isolationand IS6110-PCR in both tissues [27]

Experimental vaccination and M bovis infection wereperformed inside the biosafety BSL3 facilities for animals of


the National Institute of Agricultural Technology (INTA)Argentina in compliance with the regulations of the EthicalCommittee of INTA (CICUAE) and the biosafety protocolsas authorized by the National Service of Agricultural andFood Health and Quality (SENASA)

(2) Naturally Map M bovis-Infected Cattle and Free TBB andPTB Herd For the purpose of verifying the TBB cocktailsspecificity by IFN-120574 assay we used blood samples of 10and 17 animals from 2 dairy herds with PTB confirmed byfecal culture The selected animals were positive by DTH forPPD-A

A total of 58 animals from M bovis-infected beef herdwere 27 DTH positive (induration reaction ge 5mm) 19displayed intermediate reaction (1ndash3mm) and 12 animalswere DTH negative

As a negative control 10 animals from a free TBB and PTBherd were used for IFN-120574 release assay

24 Evaluation of C1 and C2 in BCG-Vaccinated Experiment-ally M bovis-Infected and Naturally Map-Infected Cattleby IFN-120574 Release Assay Blood samples from infected andvaccinated cattle were collected from the jugular vein intoheparinized vials at 20 30 60 and 90 days after M bovisinfection (dpi) and 30 and 60 days after BCG vaccina-tion (dpv) These samples were used to evaluate cocktailsand fractions from PPD-B by IFN-120574 release assay using acommercial ELISA-based kit (Bovigam Prionics ShlierenZurich Switzerland) Briefly aliquots of 200120583L of bloodwereadded in a 96-well culture microplate and incubated with25 120583L of antigenic preparations per duplicate Each of theconstituent proteins was added to the cocktails at a concen-tration of 55120583gmL and the PPD-B peptidic fractions wereused at a concentration of 36 120583gmL of total protein PPD-Aand PPD-B were used at a final concentration of 50120583gmLNegative-control wells with PBS alone were included foreach tested animal As positive control 45 120583gmL pokeweedmitogen (Sigma-Aldrich United Kingdom) was used Theplates were incubated in a humidified 5 CO

2incubator at

37∘C for 16 h Stimulated plasma was obtained by centrifu-gation and IFN-120574 concentrations in plasma were determinedfollowing the manufacturerrsquos procedures Color developmentwas measured at 450 nm and the results were expressed asoptical density (OD) indices (ODIs) (ODI = OD for antigensstimulated culturesOD for PBS stimulated cultures) AnODI equals or higher than 2 was considered positive Thecoefficient of variation between duplicate wells was less than5 and the OD for the control wells was usually less than 01

Also blood samples of 17 animals from a dairy herd withPTB positive to DTH with PPD-A were stimulated with C1C2 PPD-A or PPD-B to test specificity in the IFN-120574 releaseassay

25 DTH Skin Test in Experimentally M bovis-Infected CattleThe experimentally M bovis-infected animals were testedby DTH before and after infection Before infection all theanimals used in this study were DTH negative using PPD-B (lt1mm) The cattle were reevaluated by DTH 90 days

after M bovis challenge using C1 C2 and PPD-B Thecocktails for DTH were prepared with 10 120583g of each antigenper 01mL in PBS and 100 120583g per 01mL of PPD-B Animalswere intradermally injected with 01mL of C1 C2 or PPD-B (1mgmL) and the thickness of the caudal fold tuberculinskin test was measured using callipers before and 72 h afterinjection

26 Antigen Identification in PPD-B Fractions PPD-B wasprepared and fractionated in order to identify new antigenscomponents of the PPD-B capable to improve the immuno-logical cellular response in animalswithTBB BrieflyMbovisstrain AN5 was cultured in modified Dorset-Henley mediumfor 8 to 10 weeksThe cultures were inactivated by autoclave at100∘C for 3 hours and the proteins were concentrated using aPellicon XI device (Millipore Corporation Bedford MA) fortangential flow filtration

261 Separation of Protein Fractions from PPD-B PPD-Bproteins were divided into narrow molecular mass fractionsby continuous elution of polyacrylamide gels as describedpreviously [29] Briefly PPD-B preparation was resuspendedin cracking buffer (SDS 2 Tris HCl 0125M pH 68 2-mercaptoethanol 1 bromophenol blue 002 and glycerol10) heated for 5min in boiling water and resolved bysodium dodecyl sulfate-polyacrylamide gel electrophoresis12 (SDS-PAGE) Thirty fractions were collected using awhole-gel elutor (Bio-Rad Corporation) The protein con-centration of the different fractions was estimated by themicro BSA Protein Assay kit (Pierce USA) The fractionswere dialyzed against PBS and kept frozen at minus80∘C untilused

262 Identification of New Antigens from Bovine PPD Frac-tions by Mass Spectrometry PPD-B fractions were evaluatedfor their capability to stimulate IFN120574 release as previouslydescribed and the proteins present in the selected fractionswith higher ODIs were identified by mass spectrometryThe selected samples were digested with sequencing-gradetrypsin (Promega) by incubation at 35∘C overnight Prior toMS analyses the digested samples were desalted using C18reverse phase microcolumns (Omix Tips Varian) Brieflycolumns were preequilibrated with 20 120583L of an aqueoussolution of 01 trifluoroacetic acid (TFA) After sampleloading microcolumns were washed with 01 TFA andeluted with 01 TFA in 60 acetonitrile

The samples were evaporated in the speed vac resus-pended in 12 120583L of 005 formic acid and then injected ina nanoLC equipment (Proxeon easynLC Thermo Scientific)Peptide separation was performed in a reverse-phase column(easy C18 column 3120583m 75 120583m IDtimes10 cm ProxeonThermoScientific) and peptides were eluted using a 01 (vv) formicacid in water 01 (vv) formic acid in acetonitrile gradient(0ndash60 acetonitrile in 60min flow 400 nlmin) Online MSdetectionanalysis was carried out in a linear ion trap massspectrometer (LTQ Velos Thermo Fisher Scientific CorpUSA) with nanospray ionization Proteins were identified byNCBInr database searching (November 2009) with peptide


PBS PPD-A PPD-B C1 C20

10

20

30

Skin

thic

knes

s (m

m)

ESAT-6CFP-10

lowastlowastlowast

lowast

lowastlowast

(a)

ESAT-6CFP-10


10

20

Skin

thic

knes

s (m

m)

lowastlowast

lowast

lowast

lowast

lowast

lowast

(b)

Figure 1 DTH response induced by PPD-A PPD-B C1 C2 ESAT-6CFP-10 and PBS in guinea pigs previously sensitized with M avium(a) or M bovis (b) Responses were measured at 24 hs Statistical differences between responses were found by using Kruskall Wallis test( lowastlowast119875 lt 001 lowast119875 lt 005)

mz values using theMASCOT search engine (version 2302)in the MSMS ion search mode and the following searchparameters taxonomy Mycobacteriun tuberculosis complexpeptide tolerance 15 Da fragmentmass tolerance 08Da andmethionine oxidation as the allowed variable modificationSignificant protein scores and individual ion score (119875 lt 005)were used as criteria for positive protein identification

263 Evaluation of Novel Enriched C1 by IFN-120574 The per-formance of the enriched C1 with the recombinant antigensidentified from highly reactive fractions of PPD-B (novelcocktails C1 + FixB C1 + CFP2) was assessed using bloodfrom a beef herd with high prevalence of TBB (119899 58) by IFN-120574 release assay In addition we also used blood samples froma dairy herd with PTB (119899 10) and from a TBB and PTB freeherds (119899 10)

3 Results

31 Evaluation of the Biological Potency of C1 andC2 inGuineaPigs We initially assessed the application of antigens thathave been extensively tested in TBB with satisfactory resultsThese antigens were used to produce two cocktails cocktail1 (C1) containing ESAT-6 CFP-10 and MPB83 and cocktail2 (C2) with ESAT-6 CFP-10 MPB83 MPB70 TB103 andHspX The C1 and C2 were first tested in M bovis- and Mavium-sensitized guinea pigs by DTH reaction by detectionof swelling 24 hs after inoculation at the site of injection Thereactions in M bovis-sensitized guinea pigs were similar toPPD-B when C1 or C2 was injected (Figure 1(b)) HoweverinM avium-sensitized guinea pigs PPD-B had higher valuesthan C1 (Figure 1(a)) Then C1 is a potential specific reactivefor diagnosis of TBB (Figure 1) In order to evaluate ifthe addition of MPB83 to ESAT-6CFP-10 compromises thespecificity of C1 we tested ESAT-6CFP-10 without MPB83

The response was not significantly different in M avium-sensitized guinea pigs and then we could infer that theaddition of MPB83 to C1 would not compromise differentialdiagnosis

When all the antigens were assayed in a group of nonsen-sitized guinea pigs no reactionwas detected (data no shown)

32 C1 and C2 Evaluation by IFN-120574 Release in Experi-mentally M bovis-Infected BCG-Vaccinated Naturally andMap-Infected Cattle The relative amounts of IFN-120574 weremeasured stimulating blood from experimentally M bovis-infected bovines using C1 C2 PPD-A and PPD-B No sig-nificant differences were found at 30 60 and 90 dpi betweenC1 C2 and PPD-B However C1 and PPD-B stimulatedhigher values of IFN-120574 than C2 20 dpi (Figure 2(a)) In BCG-vaccinated animals (119905 = 30 and 60 dpv) no significantdifferences were detected between the three antigens How-ever prior vaccination (119905 = 0) IFN-120574 levels were above 2when blood was stimulated with PPDB (Figure 2(b)) For thepurposes of this study an ODI equals or higher than 2 wasconsidered positive

Regarding the relative amounts of IFN-120574 induced by C1C2 and PPD-B in naturally Map-infected cattle PPD-B andC2 detected 617 and 417 positive animals respectively whileC1 detected only one (Figure 3) These results demonstratedthat C1 is more specific for TBB diagnosis than C2 and PPDBsince this cocktail was effective in suppressing the detectionof animals infected with other mycobacteria

33 C1 and C2 Evaluation by DTH in Experimentally Mbovis-Infected Cattle M bovis-infected cattle were tested forDTH 90 dpi using C1 C2 and PPDB as antigens Values atthe induration area were significantly lower when injectingC1 and C2 than swelling at the site of injection of PPD-BHowever if skin test is considered as positive (values above


0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2

Time after infection (days)

OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2

Time after vaccination (days)O

DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)

Figure 2 IFN-120574 responses induced by PPD-A PPD-B C1 and C2 in experimentallyM bovis-infected cattle (119899 = 6) (a) and BCG-vaccinatedanimals (119899 = 5) (b) at different times The cocktails were tested at a concentration of 55 120583gmL per constitutive protein PPD-A and PPD-B with concentration of 50120583gmL Aliquots of 25 120583L of each antigenic preparation were added to 200 120583L of blood samples The results areexpressed as mean of ODIs with standard errors Statistical differences between responses were found by using MannWhitney test lowast differs(119875 lt 005) from PDD-A and C2 values at 20 dpi for both PPD-B and C1 + differs (119875 lt 005) from PPD-A values at 60 and 90 dpi for PPD-BC1 and C2

PPD-A PPD-B C1 C20123456789

10111213

OD

I

Figure 3 IFN-120574 responses induced by PPD-A PPD-B C1 and C2in naturally Map-infected cattle (119899 = 17) The cocktails were testedat a concentration of 55 120583gmL per constitutive protein PPD-A andPPD-B were assessed with concentration of 50120583gmL Aliquots of25 120583L of each antigenic preparation were added to 200 120583L of bloodsamples The results for each animal are represented by differentfigures and the horizontal line provides the mean of the ODIs Thedashed line represents the cutoff values used for positivity

5mm) C1 andC2 detected 56 reactors to PPD-B inM bovis-infected animals (Figure 4)

It is worth to mention that C1 and C2 were intradermallyinjected at a concentration of 10 120583g of each antigen per 01mLin PBS while PPD-B was inoculated at 100 120583g01mL Thishigher concentration could be responsible for the differencesto the reactions in response to PPDB

34 Evaluation of PPD-B Fractions by IFN-120574 in Experimen-tally M bovis-Infected and BCG-Vaccinated Animals Thirtyprotein fractions with molecular masses ranging from lt17to 90 kDa were obtained by electroelution of PPD-B in a12 SDS-PAGEThese fractions were used to stimulate bloodfrom experimentally M bovis-infected and BCG-vaccinatedcattle by IFN-120574 release assays All fractions were testedwith blood taken at 0 and 20 dpi We observed that PPD-B fractions with the lowest molecular masses were moreantigenicTherefore these samples were further checkedwithblood taken at 30 60 and 90 dpi (Figure 5) These sampleswere also used to test blood from BCG-vaccinated cattleby IFN-120574 release tests Blood was collected at 30 and 60dpv and most PPD-B fractions showed low levels of ODI(Figure 6) The fractions 21 23 and 24 were selected forprotein identification


0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast

Figure 4 DTH response induced by PPD-B C1 and C2 in exper-imentally M bovis-infected cattle to 90 dpi (119899 = 6) The cocktailswere prepared with 10120583g of each antigen per 01mL in PBS andPPD-B 100 120583g per 01mL The animals were intradermally injectedwith 01mL of C1 C2 or PPD-B (1mgmL) and the thickness ofthe caudal fold tuberculin skin test was measured using callipersprior and 72 h after injection Animal responses were representedby boxes and the horizontal line provides the median with standarderrors The statistical difference between responses was determinedby using Kruskall Wallis test ( lowastlowast119875 lt 001)

35 Protein Identification from Selected PPD-B Fractions byMass Spectrometry Selected fractions were analyzed by massspectrometry to identify individual proteins The three frac-tions were subjected to the LC-MSMS but only one fraction(number 23) allowed the identification of seven putativeantigens CFP10 CFP2 MPB70 MPB83 FixB PepA HspXand a partial sequence of an unknown protein (Table 1)CFP10 MPB70 MPB83 and HspX are known as antigenicproteins andwere already included in C1 or C2 CFP2 belongsto the CFP protein family which is the founding member ofthe family CFP10 is a putative secreted protein that may playa role in the development of protective immune responseFixB is an electron transfer flavoprotein that functions asa specific electron acceptor for other dehydrogenases PepAis a probable serine protease Finally the detected partialsequence yielded significant alignments with a possible trans-port protein SecE2 (M tuberculosisRv0379) with a sequenceidentity of 60 (70116 aa)

36 Evaluation of the Novel Recombinant Mycobacterial Pro-teins from PPD-B Fractions The genomic sequences corre-sponding to cfp2 fixb and pepa were cloned expressed andpurified as described above We first checked the specificityof the antigens by IFN-120574 in Map-infected animals CFP2

and FixB did not detect any animals with PTB while PepAdetected 2 animals in a herd with PTB (data not shown)

Based on our results C1 displayed better performancediagnostic than PPD-B and even better than C2 by IFN-120574assay indeed C1 detected animals with TBB and showed theleast detection of animals either vaccinated or infected withMap Thus we prompted to improve C1 with the addition ofthe recombinant proteins CFP2 or FixB

The novel cocktails were tested in 58 animals from aherd with TBB According to the average value of IFN-120574the response to PPD-B was similar to that of C1 with FixB(average ODI = 3) (Figure 7) PPD-B detected 3358 animalswhile C1 detected 2458 animals suspected to be infectedwith M bovis The addition of FixB to C1 improved theresults detecting 2958 animals By contrast the addition ofCFP2 did not yield better results detecting 2258 (Figure 7Table 2)

Most importantly C1 C1 plus FixB and C1 plus CFP2 didnot detect animals neither in a PTB herd nor in healthy cattle(Figures 8 and 9) Thus the inclusion of FixB in C1 enhancedthe recognition of naturally M bovis-infected cattle withoutcompromising specificity

4 Discussion

Several immunodominant proteins identified fromM tuber-culosis and M bovis have been identified by comparativegenomics [4] differential transcription rates [5] or geneexpression profiles associated with latent mycobacterialinfection [6 7] Others have been detected from members ofthe PEPPE family [8] proteins from crude protein fractionsof M bovis and potentially secreted proteins Within thesepotentially secreted proteins we canmentionmembers of theEsx family such as ESAT-6 CFP-10 and TB103 (Rv3019c)[10 11 15]

CFP-10 ESAT-6 and TB103 are members of a largefamily ofmycobacterial proteins typically consisting of about100 amino acids and are characterized by their organizationin pairs within the genome [30] Members of this familyhave been identified as potent T-cell antigens [15 31] Ourgroup has previously participated in a multilaboratory studythat assessed the sensitivity and specificity of the IFN-120574assay using several antigens in cattle naturally infected withM bovis from Northern Ireland Mexico and ArgentinaThese regions have low medium and high prevalence ofTBB respectively In the three countries ESAT-6 and CFP-10 performed as superior diagnostic antigens [16] Otherprevious studies have demonstrated that antigens ofM bovissuch asMPB70 andMPB83 also induced strong proliferationand IFN-120574 responses in vitro in M bovis-infected animalswhile BCG-vaccinated or M avium-sensitized animals didnot respond to these antigens [19]These results thus confirmthat MPB83 and MPB70 also might be suitable antigensto differentiate between animals with TBB from animalswith PTB- or BCG-vaccinated as well as from animalsvaccinated against PTB MPT83 (Rv2873) is a cell wall-associated lipoglycoprotein ofM tuberculosiswhose functionis still unknown However this protein has been suggested


0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM

Figure 5 IFN-120574 responses induced by PPD-B fractions in experimentally M bovis-infected cattle (119899 = 6) PPD-B fractions were testedat a concentration of 36 120583gmL Aliquots of 25 120583L of each fraction were added to 200 120583L of blood samples The results for each animal arerepresented by different vertical linesThe fractions were first tested with blood of animals from 0 and 20 dpiThe fractions that displayed themore stimulant responses were tested again at 30 60 and 90 dpi

02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM

Figure 6 IFN-120574 responses induced by PPD-B fractions in BCG-vaccinated cattle (119899 = 5) PPD-B fractions were tested at a concentration of36 120583gmL Aliquots of 25 120583L of each fraction were added to 200 120583L of blood samples The results for each animal are represented by differentvertical lines The fractions were tested with blood of animals taken at 0 30 and 60 dpv

Table 1 Identified antigens from PPD-B by mass spectrometry

Accession number Rv Mb Proteins Score Length (aa)gi 15611010 Rv3874 Mb3904 10 kda culture filtrate antigen EsxB or CFP10 182 100gi 149926 Rv2875 Mb2900 MPB70 135 193gi 2149409lowast Unknown 113 116gi 15609513 Rv2376c Mb2397c Low molecular weight antigen CFP2 102 168gi 15610165 Rv3028c Mb3054c Electron transfer flavoprotein subunit alpha FixB 85 318gi 15607267 Rv0125 Mb0130 Serine protease PepA 70 355

gi 248681lowastlowast MMP = 19 kDa major membrane protein [Mtuberculosis Erdman strain 107] 73 143

gi 6469702 Rv2873 Mb2898 MPB83 (Mycobacterium tuberculosis) 63 220lowastSequence producing significant alignments with the protein of M tuberculosis Rv0379 possible protein transport protein SecE2 with identities in 70 from116 aalowastlowastSequence producing alignments with HspX ofM tuberculosisM bovis


Table 2 Number of recognized naturallyM bovis infected animalsby the different antigens by IFN-120574 release assay

Antigen Number of positive animals by IFN-120574totalPPD-B 3358C1 2458C1 + FixB 2958C1 + CFP2 2258

0123456789

10111213141516171819

OD

I

PPD-A PPD-B C1 C1 + FixB C1 + CFP2

Figure 7 IFN-120574 responses induced by PPD-A PPD-B C1 C1plus FixB and C1 plus CFP2 in cattle from a herd with TBB(119899 = 58) The cocktails were tested at a concentration of 55120583gmLper constitutive protein PPD-A and PPD-B with concentration of50 120583gmL Aliquots of 25 120583L of each antigenic preparation wereadded to 200 120583L of blood samples The results for each animal arerepresented by different markers and the horizontal line providesthe mean of the ODIs The dashed line represents the cutoff valuesused for positivity

to play a role in adhesion and dissemination based on seq-uence analysis Its homologue in M bovis MBP70 is alsoa serodominant antigen during M bovis infection in cattleMPB83 and MPB70 are major antigens highly expressed byM bovis and considerably less abundantly expressed by Mtuberculosis [32ndash34]

In a prior study in our laboratory eleven proteins weredetected during the evaluation of the fractions from filtrateand cell extracts from M bovis that elicited IFN-120574 responsein animals with TBB Among the detected proteins EsxI andHspX triggered a high T cell immune response as measuredby IFN-120574 release assay [9]

According to these previous results we selected therecombinant proteins included in each cocktail Initiallycocktails composed of purified recombinant M bovisMtuberculosis antigenic proteins HspX TB103 ESAT-6 CFP-10 MPB70 and MPB83 were formulated C1 containedthe following proteins ESAT-6 CFP-10 and MPB83 andC2 contained ESAT-6 CFP-10 MPB83 HspX TB103 andMPB70

0123456789

1011121314151617181920

OD

I

C1 C1 + FixB C1 + CFP2PPD-A PPD-B

Figure 8 IFN-120574 responses induced by PPD-A PPD-B C1 C1plus FixB and C1 plus CFP2 in cattle from a herd with PTB(119899 = 10) The cocktails were tested at a concentration of 55120583gmLper constitutive protein PPD-A and PPD-B with concentration of50 120583gmL Aliquots of 25 120583L of each antigenic preparation wereadded to 200 120583L of blood samples The results for each animal arerepresented by different figures and the horizontal line provides themean of the ODIs The dashed line represents the cutoff values usedfor positivity

0

1

2

3

4

5

OD

I

PWM C2PPD-A PPD-B C1 C1 +FixB

C1 +CFP2

Figure 9 IFN-120574 responses induced by PPD-A PPD-B C1 C2 C1plus FixB and C1 plus CFP2 in cattle from a free TBB and PTB herd(119899 = 10) The cocktails were tested at a concentration of 55120583gmLper constitutive protein PPD-A and PPD-B with concentration of50 120583gmL Aliquots of 25 120583L of each antigenic preparation wereadded to 200 120583L of blood samples The results for each animal arerepresented by different markers and the horizontal line providesthe mean of the ODIs The dashed line represents the cutoff valuesused for positivity

When the potency of these cocktails was evaluated byDTH in M avium-sensitized guinea pigs C1 was more spe-cific than PPD-B C1 did not show significant differences incomparisonwith the response of CFP-10ESAT-6 then in thisexperience the inclusion ofMPB83 to C1 did not compromise


differential diagnosis However oneM avium-sensitized ani-mal showed response against protein cocktail that includedESAT-6 and CFP-10 antigens absent in M avium but thisresponse was similar to the controls inoculated with PBS

In addition when using protein-antigen combination ofESAT-6 CFP-10 and MPB83 in the IFN-120574 released assayC1 was highly sensitive and specific in the detection ofexperimentally M bovis-infected cattle This combinationelicited no response in BCG-vaccinated calves and showedthe least detection of Map-infected animals On the otherhand C2 had poor IFN-inducing capacities in experimentallyM bovis-infected cattle at 20 dpi Despite the immunogenic-ity of MPB70 Hspx and TB103 their inclusion in C2 didnot increase the IFN-120574 response compared with the use ofonly ESAT-6 CFP-10 and MPB83 at 20 30 60 and 90 dpiSince some of these antigens as ESAT-6 and CFP-10 are withTB103 members of the same family and MPB70 and MPB83are closely related sharing 73 protein sequence identityit is likely that their combination may result in antigenredundancy C1 formulated with dominant T cell antigensESAT-6 and CFP-10 also contained MPB83 B antigens suchas MPB70 and MPB83 may still have a role in promotingreaction initiation ofDTH response thereby can help to elicita better response to dominant effector antigens [13]

In the current study 56 PPD-B reactors were alsopositive to DTH skin reactions when using C1 and C2as immunogens in experimentally M bovis-infected cattleDTH response in experimentally M bovis-infected cattlewas significantly higher when injecting PPD-B than whenusing C1 or C2 This latter difference could be due to thelower concentration of each protein 10120583g in C1 and C2compared to 100 120583g of PPD-B In previous field studiesthis concentration improved skin test responses withoutcompromising specificity [13 17] The results of this studysuggest that the doses of the recombinant proteins in thecocktails C1 and C2 for tuberculin skin test may have to beoptimized

The sensitivity of the DTH is less than 80 whichmakes it unlikely as an only diagnostic tool for an efficienteradication of tuberculosis from a herd [35] Therefore inspite of the higher cost and complexity the IFN-120574 releaseassay can be used as a complementary test to the intradermalskin test to confirm or discard the first results The use ofC1 in the IFN-120574 release test demonstrated clear benefits sinceless responses were obtained when comparedwith PPD-B inBCG-vaccinated orMap-infected calves however the resultsconfirmed that the specificity of the test is compromisedwhen PPD-B is used as immunogen in cattle sensitized orinfected with other mycobacteria A minimum difference inthe performance of C1 was observed between herds withPTB (Figures 3 and 8) it could be attributed to differentenvironmental exposure Finally the addition of FixB to C1resulted in higher sensitivity than C1 when evaluated in aherd with TBB On the other hand the addition of CFP2 toC1 did not result in a significant improvement in IFN-120574 assaywith C1

Even though less animals were recognized with C1 plusFixB compared with PPD-B the novel cocktail was morespecific than PPD-B Most important in spite of the fact

that only 58 bovine with TBB were tested the inclusionof FixB in C1 enhanced the recognition of these naturallyM bovis-infected cattle without compromising specificityof IFN-120574 assay demonstrated that a C1 plus FixB couldbe suitable candidates for the development of diagnosticreagents to either differentiate betweenM bovis-infected andMAP infectedBCG-vaccinated animal and then improve thespecificity of the diagnosis of TBB

5 Conclusions

This study demonstrates that cocktails containing definedM tuberculosis complex antigens such as ESAT-6 CFP-10 MPB83 and FixB can provide a sensitive and specificdiagnosis of TBB This finding is relevant since a DIVA anda differential diagnosis between animals with TBB and PTBis needed BCG vaccination could be applied in combinationwith such DIVA tests together with PTB vaccines Theseimportant proof-of-principle data provide a basis for futureoptimization and improvement of protein concentration inthe novel cocktail

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors are grateful toDra Julia Sabio yGarcıa for criticalreading of this paper This work was funded by The NationalAgency for Science and Technology Promotion with fundsfrom Argentinean Sector Fund (FONARSEC) Marıa LauraMon Roberto DamianMoyanoMariana Noelia Viale MarıaAlejandra Colombatti Olivieri Marıa de la Paz Santangeloand Marıa Isabel Romano are CONICET fellows

References

[1] I Etchechoury G E Valencia N Morcillo et al ldquoMoleculartyping of mycobacterium bovis isolates in Argentina firstdescription of a person-to-person transmission caserdquo Zoonosesand Public Health vol 57 no 6 pp 375ndash381 2010

[2] S Borsuk J Newcombe TAMendumOADellagostin and JMcFadden ldquoIdentification of proteins from tuberculin purifiedprotein derivative (PPD) by LC-MSMSrdquo Tuberculosis vol 89no 6 pp 423ndash430 2009

[3] T S K Prasad R Verma S Kumar et al ldquoProteomic analysisof purified protein derivative of Mycobacterium tuberculosisrdquoClinical Proteomics vol 10 no 1 article 8 2013

[4] P J Cockle S V Gordon A Lalvani B M Buddle R GHewinson and H M Vordermeier ldquoIdentification of novelMycobacterium tuberculosis antigens with potential as diag-nostic reagents or subunit vaccine candidates by comparativegenomicsrdquo Infection and Immunity vol 70 no 12 pp 6996ndash7003 2002

[5] B Sidders C Pirson P J Hogarth et al ldquoScreening of highlyexpressedmycobacterial genes identifies Rv3615c as a useful dif-ferential diagnostic antigen for the Mycobacterium tuberculosis


complexrdquo Infection and Immunity vol 76 no 9 pp 3932ndash39392008

[6] G J Jones C Pirson H P Gideon et al ldquoImmune responsesto the enduring hypoxic response antigen Rv0188 are preferen-tially detected in Mycobacterium bovis infected cattle with lowpathologyrdquo PLoS ONE vol 6 no 6 Article ID e21371 2011

[7] H PGideonKAWilkinson T R Rustad et al ldquoBioinformaticand empirical analysis of novel hypoxia-inducible targets of thehuman antituberculosis T cell responserdquo Journal of Immunol-ogy vol 189 no 12 pp 5867ndash5876 2012

[8] H M Vordermeier R G Hewinson R J Wilkinson et alldquoConserved immune recognition hierarchy of mycobacterialPEPPE proteins during infection in natural hostsrdquo PLoS ONEvol 7 no 8 Article ID e40890 2012

[9] V Meikle A Alito A S Llera et al ldquoIdentification of novelMycobacterium bovis antigens by dissection of crude proteinfractionsrdquo Clinical and Vaccine Immunology vol 16 no 9 pp1352ndash1359 2009

[10] A Alito J McNair R M Girvin et al ldquoIdentification ofMyco-bacterium bovis antigens by analysis of bovine T-cell responsesafter infection with a virulent strainrdquo Brazilian Journal ofMedical and Biological Research vol 36 no 11 pp 1523ndash15312003

[11] J M Pollock J McNair H Bassett et al ldquoSpecific delayed-typehypersensitivity responses to ESAT-6 identify tuberculosis-infected cattlerdquo Journal of Clinical Microbiology vol 41 no 5pp 1856ndash1860 2003

[12] A O Whelan J C Hope C J Howard D Clifford R GHewinson and H M Vordermeier ldquoModulation of the bovinedelayed-type hypersensitivity responses to defined mycobacte-rial antigens by a synthetic bacterial lipopeptiderdquo Infection andImmunity vol 71 no 11 pp 6420ndash6425 2003

[13] A O Whelan D Clifford B Upadhyay et al ldquoDevelopmentof a skin test for bovine tuberculosis for differentiating infectedfrom vaccinated animalsrdquo Journal of Clinical Microbiology vol48 no 9 pp 3176ndash3181 2010

[14] J M Pollock and P Andersen ldquoThe potential of the ESAT-6antigen secreted by virulent mycobacteria for specific diagnosisof tuberculosisrdquo Journal of Infectious Diseases vol 175 no 5 pp1251ndash1254 1997

[15] P J Cockle S V Gordon R G Hewinson and H M Vorder-meier ldquoField evaluation of a novel differential diagnosticreagent for detection ofMycobacterium bovis in cattlerdquo Clinicaland Vaccine Immunology vol 13 no 10 pp 1119ndash1124 2006

[16] C Aagaard M Govaerts V Meikle et al ldquoOptimizing antigencocktails for detection of Mycobacterium bovis in herds withdifferent prevalences of bovine tuberculosis ESAT6-CFP10mixture shows optimal sensitivity and specificityrdquo Journal ofClinical Microbiology vol 44 no 12 pp 4326ndash4335 2006

[17] S Flores-Villalva F Suarez-Guemes C Espitia A O WhelanM Vordermeier and J A Gutierrez-Pabello ldquoSpecificity of thetuberculin skin test is modified by use of a protein cocktailcontaining eSAT-6 and CFP-10 in cattle naturally infected withMycobacterium bovisrdquoClinical and Vaccine Immunology vol 19no 5 pp 797ndash803 2012

[18] C Casal J Bezos A Dıez-Guerrier et al ldquoEvaluation oftwo cocktails containing ESAT-6 CFP-10 and Rv-3615c in theintradermal test and the interferon-120574 assay for diagnosis ofbovine tuberculosisrdquo Preventive Veterinary Medicine vol 105no 1-2 pp 149ndash154 2012

[19] H M Vordermeier P C Cockle A Whelan et al ldquoDevelop-ment of diagnostic reagents to differentiate between Mycobac-terium bovis BCG vaccination andM bovis infection in cattlerdquoClinical andDiagnostic Laboratory Immunology vol 6 no 5 pp675ndash682 1999

[20] M Vordermeier S V Gordon and R G Hewinson ldquoMycobac-terium bovis antigens for the differential diagnosis of vaccinatedand infected cattlerdquoVeterinaryMicrobiology vol 151 no 1-2 pp8ndash13 2011

[21] H M Vordermeier A Whelan P J Cockle L Farrant NPalmer and R G Hewinson ldquoUse of synthetic peptides derivedfrom the antigens ESAT-6 and CFP-10 for differential diagnosisof bovine tuberculosis in cattlerdquo Clinical and Diagnostic Labo-ratory Immunology vol 8 no 3 pp 571ndash578 2001

[22] W R Waters B J Nonnecke M V Palmer et al ldquoUse ofrecombinant ESAT-6CFP-10 fusion protein for differentiationof infections of cattle byMycobacterium bovis and byM aviumsubsp avium and M avium subsp paratuberculosisrdquo Clinicaland Diagnostic Laboratory Immunology vol 11 no 4 pp 729ndash735 2004

[23] G J Jones B L Khatri M C Garcıa-Pelayo et al ldquoDevel-opment of an unbiased antigen-mining approach to identifynovel vaccine antigens and diagnostic reagents for bovinetuberculosisrdquo Clinical and Vaccine Immunology vol 20 no 11pp 1675ndash1682 2013

[24] M Malaghini V Thomaz-Soccol C M Probst et al ldquoRecom-binant antigen production for assays of intradermoreaction fordiagnosis and surveillance of tuberculosisrdquo Journal of Biotech-nology vol 156 no 1 pp 56ndash58 2011

[25] L A H van Pinxteren P Ravn E M Agger J Pollockand P Andersen ldquoDiagnosis of tuberculosis based on the twospecific antigens ESAT-6 and CFP10rdquo Clinical and DiagnosticLaboratory Immunology vol 7 no 2 pp 155ndash160 2000

[26] KWeldingh andPAndersen ldquoESAT-6CFP10 skin test predictsdisease inM tuberculosis-infectedGuinea pigsrdquo PLoSONE vol3 no 4 Article ID e1978 2008

[27] F C Blanco M V Bianco S Garbaccio et al ldquoMycobacteriumbovis Δmce2 double deletion mutant protects cattle againstchallenge with virulentM bovisrdquo Tuberculosis vol 93 no 3 pp363ndash372 2013

[28] CNishibeA BCanevari Castelaob RDallaCosta et al ldquoDraftgenome sequence of Mycobacterium bovis 04-303 a highlyvirulent strain from Argentinardquo Genome Announcements vol1 no 6 Article ID 00931-13 2013

[29] J M Pollock and P Andersen ldquoPredominant recognitionof the ESAT-6 protein in the first phase of infection withMycobacterium bovis in cattlerdquo Infection and Immunity vol 65no 7 pp 2587ndash2592 1997

[30] K L Lightbody D Ilghari L C Waters et al ldquoMolecular fea-tures governing the stability and specificity of functional com-plex formation by Mycobacterium tuberculosis CFP-10ESAT-6family proteinsrdquo The Journal of Biological Chemistry vol 283no 25 pp 17681ndash17690 2008

[31] G J Jones S V Gordon R G Hewinson and H M Vorder-meier ldquoScreening of predicted secreted antigens fromMycobac-terium bovis reveals the immunodominance of the ESAT-6protein familyrdquo Infection and Immunity vol 78 no 3 pp 1326ndash1332 2010


[32] H G Wiker K P Lyashchenko A M Aksoy et al ldquoImmuno-chemical characterization of the MPB7080 and MPB83 pro-teins of Mycobacterium bovisrdquo Infection and Immunity vol 66no 4 pp 1445ndash1452 1998

[33] S Lesellier L Corner E Costello et al ldquoAntigen specific immu-nological responses of badgers (Meles meles) experimentallyinfected with Mycobacterium bovisrdquo Veterinary Immunologyand Immunopathology vol 122 no 1-2 pp 35ndash45 2008

[34] R G Hewinson S L Michell W P Russell R A Mcadamand W R Jacobs Jr ldquoMolecular characterization of MPT83 aseroreactive antigen ofMycobacterium tuberculosiswith homol-ogy to MPT70rdquo Scandinavian Journal of Immunology vol 43no 5 pp 490ndash499 1996

[35] J M Pollock B M Buddle and P Andersen ldquoTowardsmore accurate diagnosis of bovine tuberculosis using definedantigensrdquo Tuberculosis vol 81 no 1-2 pp 65ndash69 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


evaluate reagents with potential for TBB diagnosis Diseasecontrol can also be facilitated by using the interferon-gamma(IFN-120574) assay In their original form these tests DTH andIFN-120574 are based on the response against PPD-BThis reagentis a mix of proteins lipids and carbohydrates obtainedfrom heat-killed cultures of M bovis strain AN5 whichcompromise specificity because some of its antigenic com-ponents are present in others nonpathogenic and pathogenicmycobacteria [2 3] Animals infected with Mycobacteriumavium subsp paratuberculosis (Map) the etiologic agentof paratuberculosis (PTB) or vaccinated with BCG havedemonstrated positive response in these assays using PPD-B The specificity of these tests can be increased with the useof definedM bovis antigens

Many mycobacterial proteins have been isolated andeval-uated in DTH and IFN-120574 assays in animals with TBB[4ndash13] For instance ESAT-6 (Rv3875 or EsxA) is one of themajor antigenic targets identified in both cattle and humanswith tuberculosis [14] Several other antigens with molecularmasses of around 10 kDa and belonging to the Esx family ofproteins have been identified subsequently such as CFP10(RV3874 or EsxB) and TB103 (Rv3019c or EsxR) and theyhave been shown to be equally well recognized by T cells[15] ESAT-6 and CFP-10 or combinations of these antigenshave been extensively tested by DTH and IFN-120574 assays innaturally M bovis-infected cattle with satisfactory results[16ndash19] The studies performed in M bovis-infected cattleyielded promising results with cocktails containing ESAT-6CFP-10 plus the addition of other antigens [18ndash20] ESAT-6 and CFP-10 are encoded in a genomic region RD1 thatis absent in BCG Pasteur and thus are useful as diagnostictools for differentiating TB-infected from BCG-vaccinatedanimals (DIVA) [19] Proliferative immune response withsynthetic peptides derived from the sequences of CFP-10and ESAT-6 showed that BCG-vaccinated cattle did notrespond to this peptide cocktail whilst they all responded toPPD-B [21]

Furthermore the use of a recombinant ESAT-6 CFP-10fusion protein was useful to differentiate M bovis-infectedfrom those Map-infected cattle [22] Several studies haveidentified immunogenic proteins present in bothM bovisMtuberculosis [4 9 10 15 17] A disadvantage of those studieshowever is that they are restricted to a relatively small setof proteins biased by the initial selection criteria Recentlya nonbiased approach with an extensive library of clonesexpressing mycobacterial proteins was used and 33 geneproducts that induced IFN-120574 production in whole-bloodcultures from TB-reactor animals were detected One ofthese proteins did not induce responses in BCG-vaccinatedanimals which is suitable for DIVA diagnostic Greaterresponder frequencies were observed for BCG-vaccinatedcattle than for TB reactors with the remaining antigenswhich leads to speculate that other ldquoless immunodominantrdquoantigens maybe recognized to a greater extent in BCG-vaccinated animals [23]

These recent studies show that the search of otherantigens for a more sensitive and specific diagnosis of TBis still relevant Unfortunately few studies have shown thefeasibility ofM bovis antigens to increase specificity of DTH

using either the guinea pigmodel or experimentallyM bovis-infected cattle [24ndash26] Moreover even more informationof the use of cocktails with M bovis antigens in naturallyinfected cattle remains scarce

The present study aimed to assess cocktails containingpreviously defined antigens of M bovis in order to differ-entiate animals with TBB from vaccinated- or Map-infectedanimals For this purposewe used different approachesDTHin the guinea pig model and both DTH and IFN-120574 assays inthe cattle model The specificity of the defined antigens wasevaluated in both BCG-vaccinated and Map-infected cattleFinally novel cocktails enriched with immunogenic proteinsidentified bymass spectrometry analysis of selected antigenicfractions of PPD-B were evaluated in TBB PTB and freeherds

2 Material and Methods

21 Antigens In the first part of the present study we assessedthe application of antigens that have been extensively testedin TBB with satisfactory results These antigens were usedto produce two cocktails cocktail 1 (C1) containing ESAT-6 CFP-10 and MPB83 and cocktail 2 (C2) with ESAT-6CFP-10 MPB83 MPB70 TB103 and HspX The potency ofthe cocktails was evaluated by DTH in M bovis M aviumsensitized-guinea pigs and nonsensitized guinea pigs as wellas in experimentallyM bovis-infected cattle (DTH and INF-120574) BCG-vaccinated naturally Map-infected cattle and in afree TBB and Map herd by IFN-120574

The purified recombinant proteins ESAT-6 CFP-10MPB70 MPB83 TB103 and HspX were kindly provided byProfessor Mahavir Singh (Lionex Diagnostics andTherapeu-ticsGmbHGermany) In all cases the endotoxin content waslt127 IUmg

In the second part we characterized immunodominantantigens from the PPD-B The coding sequences ofCFP2 FixB and PepA were amplified by PCR using thefollowing primers CFP2-fw 51015840-cgcggatccatgaagatggtgaaatcga-31015840(BamHI site) and CFP2-rev 51015840- ataagctttcagttccctgcggcctgc-31015840(HindIII site) FixB-fw 51015840-cgggatccatggctgaagtactgg-31015840(BamHI site) and FixB-rev 51015840-taagcttctagcccttgcgggcc-31015840 (HindIII site) PepA-fw 51015840-aaggatccatgagcaattcgcgccg-31015840(BamHI site) and PepA-rev 51015840-ataagctttcaggccgggggtccct-31015840(HindIII site) M bovis DNA was employed as templatefor the amplifications The amplification programme wasas follows an initial step of 95∘C for 10min 35 cycles of95∘C for 1min 58∘C for 1min and 72∘C for 1min followedby a final termination step of 72∘C for 8min The completeopen reading frames of CFP2 (507 pb) FixB (957 pb) andPepA (1068) were subsequently cloned into the expressionvector pRSET-A (Invitrogen BV Leek The Netherlands)The derived constructions were transformed into Escherichiacoli BL21 (pLys) competent cells A 3-mL inoculum of E coliBL21 harboring pRSET-A with the gene inserts was diluted120 and grown to the mid-logarithmic growth phase(OD of 06) in LB-ampicillin for 16 h at 37∘C One mMconcentration of isopropyl-D-thiogalactopyranoside (IPTG)(Merck Germany) was added to induce recombinant gene











23 Animal Models



232 Cattle











2incubator at











10

20

30

Skin

thic

knes

s (m

m)

ESAT-6CFP-10

lowastlowastlowast

lowast

lowastlowast

(a)

ESAT-6CFP-10


10

20

Skin

thic

knes

s (m

m)

lowastlowast

lowast

lowast

lowast

lowast

lowast

(b)




3 Results








0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2


OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2


DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)


PPD-A PPD-B C1 C20123456789

10111213

OD

I






0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























23 Animal Models



232 Cattle











2incubator at











10

20

30

Skin

thic

knes

s (m

m)

ESAT-6CFP-10

lowastlowastlowast

lowast

lowastlowast

(a)

ESAT-6CFP-10


10

20

Skin

thic

knes

s (m

m)

lowastlowast

lowast

lowast

lowast

lowast

lowast

(b)




3 Results








0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2


OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2


DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)


PPD-A PPD-B C1 C20123456789

10111213

OD

I






0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2incubator at











10

20

30

Skin

thic

knes

s (m

m)

ESAT-6CFP-10

lowastlowastlowast

lowast

lowastlowast

(a)

ESAT-6CFP-10


10

20

Skin

thic

knes

s (m

m)

lowastlowast

lowast

lowast

lowast

lowast

lowast

(b)




3 Results








0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2


OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2


DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)


PPD-A PPD-B C1 C20123456789

10111213

OD

I






0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10

20

30

Skin

thic

knes

s (m

m)

ESAT-6CFP-10

lowastlowastlowast

lowast

lowastlowast

(a)

ESAT-6CFP-10


10

20

Skin

thic

knes

s (m

m)

lowastlowast

lowast

lowast

lowast

lowast

lowast

(b)




3 Results








0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2


OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2


DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)


PPD-A PPD-B C1 C20123456789

10111213

OD

I






0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 20 30 60 9000

25

50

75

100

125

150

PPD-APPD-B

C1C2


OD

I

lowast

+

+

(a)

PPD-APPD-B

C1C2


DI

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

(b)


PPD-A PPD-B C1 C20123456789

10111213

OD

I






0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

PPD-BC1C2

Skin

thic

knes

s (m

m)

lowastlowast

lowastlowast








4 Discussion




0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

12

14

Fraction

OD

I

0dpi20dpi30dpi

60dpi90dpi

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM


02468

101214

Fraction

OD

I

0dpi30dpv60dpv

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

30

PPD

-APP

D-B

PWM









0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0123456789

10111213141516171819

OD

I






0123456789

1011121314151617181920

OD

I



0

1

2

3

4

5

OD

I


C1 +CFP2










5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusions




Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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