Post-mortem examination and laboratory-based analysis for the diagnosis of bovine tuberculosis among dairy cattle in Ecuador

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ContentslistsavailableatScienceDirect

Preventive

Veterinary

Medicine

jo u r n al h om ep a ge :w ww . e l s e v ie r . c o m / l o c a t e / p r e ve t m e d

Post-mortem

examination

and

laboratory-based

analysis

for

the

diagnosis

of

bovine

tuberculosis

among

dairy

cattle

in

Ecuador

Freddy

Proa ˜

no-Pérez

a,b,1

_,

_Washington

_{Benitez-Ortiz}

a,b,c,2

_,

_Daniel

_Desmecht

d,3

_,

Marco

Coral

c,4

_,

_Julio

_Ortiz

c,4

_,

_Lenin

_Ron

a,5

_,

_Franc¸

_oise

_Portaels

e,6

_,

_Leen

_Rigouts

e,f,7

_,

Annick

Linden

b,∗,8

a_{International}_Centre_for_Zoonoses,_Central_University_of_Ecuador,_Quito,_Ecuador

b_Department_of_Infectious_and_Parasitic_Diseases,_Faculty_of_Veterinary_Medicine,_University_of_Liege,_Belgium c_Faculty_of_Veterinary_Medicine,_Central_University_of_Ecuador,_Quito,_Ecuador

d_Department_of_Morphology_and_Pathology,_Faculty_of_Veterinary_Medicine,_University_of_Liege,_Belgium e_{Mycobacteriology}_Unit,_Department_of_{Microbiology,}_Institute_of_Tropical_Medicine,_Antwerp,_Belgium f_Department_of_{Pharmaceutical,}_Veterinary_and_Biomedical_Sciences,_University_of_Antwerp,_Antwerp,_Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6July2010

Receivedinrevisedform26April2011 Accepted29April2011 Keywords: Veterinaryinspection Invitroculture PCR Mycobacteriumbovis

a

b

s

t

r

a

c

t

Veterinaryinspectioninslaughterhousesallowsforthedetectionofmacroscopiclesions reminiscentofbovinetuberculosis,butthepresenceofMycobacteriumbovismustbe con-firmedbylaboratorymethods.Thisstudyaimedatcomparingtheperformancesofthe standarddiagnostictoolsusedtoidentifyM.bovisintissuespecimenssampledfrom sus-piciousanimals.Duringatwoyearsperiod,1390cattlewereinspectedattheMachachi abattoirintheMejiacanton–Ecuador.Atotalof33animalswithgranulomatouslesions weredetected,representing2.33%(16/687)and2.42%(17/703)animalsexaminedin2007 and2008,respectively.Ninety-fourtissuespecimensweresampledandscreenedforthe presenceofmycobacteria.Acid-fastbacilliwereidentifiedinonethirdofthesuspicious cat-tle(11/33)andsuggestivemicroscopiclesionsin27.3%(9/33)ofthesamplesexaminedby directmicroscopyandhistopathology,respectively.CulturingonStonebrinkmediumand 16S-rRNA-basedpolymerasechainreaction(PCR)yielded36.4%(12/33)and27.3%(9/33) ofpositives,respectively.Comparedtoculture,otherdiagnosticproceduresdisplayeda lowersensitivity,with56.5%forPCR,and43.5%fordirectmicroscopyand histopathol-ogy;however,thespecificitywashigher(94.4%forPCRandmicroscopy,and97.2%for histopathology).Weconcludethatreliablepost-mortemlaboratorytestingeitherrequires thecombinationofasetofavailablediagnostictoolsornecessitatesthedevelopmentof improvednew-generationtoolswithbettersensitivityandspecificitycharacteristics.

∗ Correspondingauthorat:DepartmentofInfectiousandParasitic Dis-eases,FacultyofVeterinaryMedicine,UniversityofLiege,SartTilman20, Bât.B43a,B-4000Liège,Belgium.Tel.:+3243664051;fax:+324366 4565.

E-mailaddresses:fproano-ciz@ac.uce.edu.ec, ciz-rectoradouc@andinanet.net(F.Proa ˜no-Pérez), wbenitez-ciz@ac.uce.edu.ec(W.Benitez-Ortiz),

daniel.desmecht@ulg.ac.be(D.Desmecht),chuckcora@hotmail.com (M. Coral),julioortiz88@hotmail.com(J.Ortiz), lron-ciz@ac.uce.edu.ec

(L. Ron), fportaels@itg.be (F. Portaels), lrigouts@itg.be (L. Rigouts), a.linden@ulg.ac.be(A.Linden).

1 _{Participation:}_conception_and_design_of_the_study,_Executor_of_ﬁeld

survey,analysisofdata,draftingthepaper.Tel./fax:+59322904801.

2 _{Participation:}_conception_and_design_of_the_ﬁeld_study,_analysis_of

data,criticalreviewandcommentingondraft.Tel./fax:+59322904801.

3 _{Participation:}_laboratory_work,_critical_review_&_revision_and

com-mentingondraft.Tel.:+3243664051;fax:+3243664565.

4 _{Participation:}_ﬁeld_study._Tel./fax:₊₅₉₃₂_2904801.

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1. Introduction

Bovinetuberculosis(BTB)ispresentinmost

develop-ingcountrieswheresurveillanceandcontrolactivitiesare

notorinadequatelyimplemented(Cosivietal.,1998).In

Ecuador, there is no BTB control program in place. An

apparentprevalenceof7.95%wasobservedamongdairy

cattlefromlargeherdsin2004(Proa ˜no-Perezetal.,2006),

and it increasedto8.63% three years later in thesame

region(Proa ˜no-Pérezetal.,2009).Herdsizewas

identi-ﬁedasanimportantriskfactorinthishusbandrysystem,

andthenumberofskin-test-positivecasesalsoincreased

signiﬁcantlywithcattleage(Proa ˜no-Pérezetal.,2009).

BTBcontrolprogramsconsistofpasteurizationofmilk,

meathygienecontrolthroughmacroscopicinspectionat

slaughter(veterinaryinspection),andskintestingtodetect

andcullinfectedanimals(Collins,2006).

CattlearemainlyinfectedwithM.bovisthrough

inhala-tionofaerosolizeddroplets(GoodchildandClifton-Hadley,

2001).However,theprocessbywhichexposureto

infec-tioncanleadtoarangeofdiseaseoutcomesisnotfully

understood(Neilletal.,1994;Pollocketal.,2006).Notall

infectedcattledisplaygross lesions,but ifpresent,they

areoftenlocatedinthoraciclymphnodes(Whippleetal.,

1996)eveninapparentlyhealthycattle.Lesionsdueto

non-tuberculousmycobacteria(NTM)canbeeasilymistaken

forBTBlesions(Oloyaetal.,2007).Consequently,

labora-toryapproachesareimportanttoconﬁrmandidentifythe

mycobacterialspeciesinvolved.

Differentmethodscanbeusedtoidentifythediseasein

humansandanimals.Microscopicdetectionof

mycobac-teriaisinsensitiveanddoesnotpermittheidentiﬁcation

ofthemycobacterialspeciesinvolved.CultureofM.bovis

canbemoresensitiveand allowsfor species

identiﬁca-tion,butistimeconsumingduetotheslowgrowthofthe

bacilli(Mangiapanetal.,1996).Polymerasechainreaction

(PCR)canreducethetimefordetectionandalsoidentify

thespecies.

Theaimofthisstudywastocomparecurrentlyavailable

laboratorytools todiagnoseBTBusingabattoir samples

takenduringveterinaryinspection,characterizethe

distri-butionoflesionsobservedintheorgansofinspectedcattle,

andstudythetrueprevalenceofBTB.

2. Materialsandmethods

2.1. Studydesign

Veterinaryinspectionwascarriedoutin1390

slaugh-teredcattlefrom112differentdairyfarmsattheabattoir

of Machachi located in the Mejia canton (0◦27SL and

78◦25WL),locatedin themajordairy cattleproduction

5 _{Participation:}_analysis_of_data,_critical_review_and_commenting_on

draft.Tel./fax:+59322904801.

6 _{Participation:}_critical_review_and_revision_and_commenting_on_draft.

Tel.:+3232476551.

7 _{Participation:}_conception_and_design_of_the_study,_analysis_of_data,

criticalreviewandrevisionandcommentingondraft.Tel.:+3232476551.

8 _{Participation:}_conception_and_design_of_the_study,_analysis_of_data,

criticalreview&revisionandcommentingondraft.

area in northernEcuador. There wasnoprevious

stud-iesonBTBinthisabattoir.Intotal,29interventionswere

performedattheslaughterhouseduringtheperiodfrom

FebruarytoMarchintheyears2007and2008.Detailed

post-mortemexaminationwasperformedin687and703

cattle,respectively.Inthisstudy,allcattlewereinspected

duringeachintervention,andcattleoriginatingfromareas

outsidetheMejiacantonwereexcluded.Individualdata

wererecordedforeachanimalbytheuseofaquestionnaire

witha focusonage,breed,sex,nameoftheowner,

ori-ginoftheanimalandlesionsfoundduringtheinspection.

Fiveoftheanimalsexaminedduringtheabattoirsurvey

werepreviouslyidentiﬁedasbovinecomparative

intrader-maltuberculintest(CITT)reactorsinasurveyamongdairy

cattleintheMejiacanton(Proa ˜no-Pérezetal.,2009).

2.2. Post-mortemexamination

Detailedveterinary inspectionwascarried outonall

cattle. The lungs, liver, spleen, kidney and mammary

glandwerepalpatedcarefullyandinspectedexternallyand

internally.Mandibular,retropharyngeal,tracheobronchial,

mediastinal, hepatic, mesenteric and supramammary

lymphnodeswereslicedintothinsections(2–3mm)and

inspectedinsitufordetectionofvisiblelesions.

2.3. Samples

Tissue specimens were taken from multiple organs

usingdisinfectedknives(i.e.,chlorine10%)fromall

sus-pectedanimalsforfurtherlaboratory-baseddiagnosis.We

includedallorgans withvisiblelesions,andadded lung

tissue takenfromtheinferiorlobeinallsampledcattle,

evenifnomacroscopiclesionswerevisible.Intotal,94

tis-suesampleswerecollectedfrom33suspectedcattle(2–8

specimens/animal,dependingonthelesionsfound);65of

themweresampledfromtissueswithvisiblegrosslesions

suggestiveformycobacterialinfection,andtherestwere

sampleswithoutlesionstakenfromlungs.

Allsampleswerecollectedinindividualsteriletubes,

storedinacoolbox,andtransportedthesamedaytothe

LaboratoryofMicrobiologyattheInternationalCentreof

Zoonoses, in Quito.Upon arrival, processingof samples

was carried out usingaseptic techniques in a biosafety

cabinet to avoid cross-contamination betweensamples.

In order to perform microscopy, PCR, and in vitro

cul-ture, approximately5g of tissue were stored at −20◦_C

in 2mLEppendorf® _tubes _containing _Dubos _broth_(No.

0385-17-6,DifcoLaboratories,Detroit,USA)supplemented

withPANTA(BactecPANTApluskitNo.4404764;Becton

Dickinson,NJ,USA)toinhibitthegrowthofcontaminants

and to preserve the mycobacteria. During the

interna-tionaltransport toBelgiumthesampleswerepackedin

acooler.Allsamplesunderwent2freeze–thawcyclesdue

tothestorageandshippingpriortotheirﬁnalprocessing

at the Mycobacteriology Unit, in theInstitute of

Tropi-calMedicineofAntwerp,Belgium.Duplicatesamplesthat

werestored inEcuadorhad onlyonefreeze–thawcycle

andwereprocessedlocallyoncetheculturetechniquewas

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Inaddition,about1cm3_of_each_sample_was_stored_in

10%formalinforhistopathologicanalysesatthe

Depart-ment ofMorphology andPathology fromtheFaculty of

VeterinaryMedicineintheUniversityofLiege,Belgium.

2.4. Microscopyandinvitroculture

UponarrivalinAntwerp,allsamplesforbacteriological

examinationwerecutandhomogenizedinasterile

mor-tarwith2mLofsterilephosphatebufferedsaline.Invitro

culturewascarriedoutafterdecontaminationusingthe

“reversed”Petroffmethod(PalominoandPortaels,1998):

20minincubationwith3mLof1NHClfollowedby

neutral-isationwith3mLof1NNaOHandcentrifugationfor20min

at3000×g.Thepelletwasmixedwith2mLsteriledistilled

waterandinoculatedontoLöwenstein–Jensen(LJ)(Parks,

1997),andStonebrinkmedium(i.e.LJwithoutglycerolbut

supplementedwith0.4%sodiumpyruvate).Isolateswere

identiﬁedasM.bovisbyspoligotypingcharacterizedbya

typicallackofspacers3,9,16and37–42(Kamerbeeketal.,

1997). For Ziehl–Neelsen (ZN) staining and subsequent

microscopicinspectiontodetectacid-fastbacilli(AFB),a

slidewasprepared using1drop oftheprocessed

spec-imen.Stainedslideswereexaminedwithanormal light

microscopeusingobjective100×andimmersionoil.

2.5. Polymerasechainreaction

DNAwasextractedfromthere-suspendedpelletusing

the method developed by Mangiapan and colleagues

(1996).Aroutinely-applied,nestedPCRtargetingthe16S

ribosomalRNAgenewasperformedtodetectDNAspeciﬁc

fortheM.tuberculosis-complexusingthefollowingprimers

(Portaelsetal.,1996):P1(5

-TGCTTAACACATGCAAGTCG-3)andP2(5-TCTCTAGACGCGTCCTGTGC-3)fortheﬁrst

run,andP3(5-AACCCGGACCTTCGTCGATG-3)withP9(5

-CATGTCTTGTGGTGGAAAGCGC-3)forthesecondrun.

Sam-pleswereconsideredpositivetobovinetuberculosiswhen

abandof500bpwasobservedona2%(w/v)agarosegel.

2.6. Histopathology

Slides forhistopathologyexaminationwereprepared

using standard techniques, and processed for standard

hematoxylin–eosin (HE)and ZN staining.A samplewas

consideredpositiveiflesionscharacteristicforBTBwere

demonstrated (granulomatous inﬂammation associated

withfocalcaseousnecrosisormineralization), orifAFB

wereobservedintheZNstain(Whippleetal.,1996).

2.7. Statisticalanalysis

The true prevalence (TP) based on detection of

gross visible lesions was calculated using the

Rogan-Gladen’s Equation (1978) under a Bayesian modelling

approaches (Berkvens et al., 2006). The Rogan-Gladen

equation describes the estimation of TP through the

apparent prevalence (P), and the Se, and Sp of a test

[P=TPSe+(1−TP)(1−Sp)].Sincethereisnogoldstandard

test, TP mustbe estimatedimposing constraintsonthe

parameters(Berkvensetal.,2006).Bayesianapproachlets

toincorporateexternalinformationbyspecifyingprior

dis-tributionsontheparameters,i.e.,priorknowledgeand/or

beliefsregardingtoSeandSpofnecropsy(Enoeetal.,2000;

Branscumetal.,2005).Thisapproachmakespossible

infer-encesaboutSeandSpwith95%probabilityinterval(95%PI)

ofnecropsyusedinthisstudy.ThepriordistributiononSe

waselicitedfromapreviousstudythatconsidersthemost

likelyvalue(modal),thustheSeofnecropsywas0.8with

a10thpercentileof0.55;whichisacceptableforthistest

andwaschosenbecauseasimilarprocedureduringthe

necropsywasperformedinthesamelymphnodes(Norby

et al.,2004), and basedonthe samemeasuresthrough

veterinaryinspectiontodiscardcarcassesinEcuador

(Per-sonal communication).Theprior distribution forthe Sp

waselicitedfromtheexperienceofveterinarianinspectors

fromtheslaughterhouseoftheMejiacanton,takinginto

accountthepresenceofotherinfectiousdiseasescausing

lesionsin lymph nodes specially in dairy cattle, thus a

modalvalueof0.95witha5thpercentileof0.80wereset

tomodeltheuncertaintyaboutSp.TheBetaBustersoftware

(availableat http://www.epi.ucdavis.edu/diagnostictest/)

(Branscum et al., 2004) computed the two parameters

ofaBetapriordistributionsbasedonthemodeandone

percentileforSeandSp,i.e.,BetaforSe(5.28,2.07),and

BetaforSp(21.20,2.06).Abinomialmodelwasbuiltand

Bayesian estimation was done through Gibbs sampling

inWinBUGS(Spiegelhalteretal.,1996).Aburn-inphase

of 1000 iterations was used and the model was run

for another 10,000 iterations to obtain estimates. The

outcomesweremeanandpercentiles (i.e.,2.5and97.5)

sampledfromtheposteriordistributionsofTP,SeandSp.

Threechainsstartingindifferentvaluesweresetandtheir

convergencewasanalyzedthroughtraceplot.Additionally

theeffectofdifferentpriordistributionsfortheparameters

ofthemodelwasanalyzedinordertoevaluatehowprior

beliefscouldaffecttheposteriorestimatesofTP,Seand

Sp.Seven scenarios were builtassuming prior different

beliefs abouttheparameters (Table1): (Scenario 1)No

priorconstraints:TP∼Uniform(0,1),Se∼Uniform(0,1)

and Sp∼Uniform (0,1);(Scenario 2) Priordistributions

forSe and Spuniformly distributedaccording toranges

suggested by Norby et al. (2004), and expert opinion

of veterinaries in slaughterhouses TP_∼Uniform (0, 1),

Se∼Uniform (0.51, 0.98) and Sp∼Uniform (0.80, 0.90);

(Scenario 3) True prevalence constrained uniformly to

valueslessthat10%andSeandSpwithsimilarvaluesto

previousexperiencesTP∼Uniform(0,0.10),Se∼Uniform

(0.50,1.00)andSp∼Uniform(0.70,1.00);(Scenario4)True

prevalenceconstraineduniformly tovaluesless that 5%

andSeandSpwithsimilarvaluestopreviousexperiences

but widen: TP∼Uniform (0, 0.05), Se∼Uniform (0.40,

1.00)andSp∼Uniform(0.60,1.00);(Scenario5)Assuming

thatSeofthenecropsyis70%andSp80%butwithhigh

uncertainty TP∼Uniform (0,1.00),Se∼Beta (8.00, 4.00)

andSp∼Uniform(9.00,2.00);Scenario6)AssumingthatSe

ofthenecropsyis70%andSp80%butwithlowuncertainty

TP∼Uniform(0,1),Se∼Beta(71.0,31.0)andSp∼Uniform

(86.0,16.0);and ﬁnally(Scenario7)TP∼Uniform(0,1),

Beta(5.28,2.07)andBeta(21.20,2.06)whichcorrespond

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Table1

EstimatesofparametersintheBayesianbinomialmodelusingdifferentpriordistributionsforTP,SeandSpofnecropsytoidentifybovinetuberculosis.

Scenario Priordistribution Posteriormean% IP95%

Scenario1 TP∼Uniform(0,1) Didnotconverge –

Se∼Uniform(0,1) Didnotconverge –

Sp∼Uniform(0,1) Didnotconverge –

Scenario2 TP∼Uniform(0,1) 1.11 0.005–4.69 Se∼ Uniform(0.51,0.98) 71.89 68.80–94.60 Sp∼ Uniform(0.80,0.90) 98.22 98.08–99.64 Scenario3 TP∼Uniform(0,0.10) 1.83 0.008–4.40 Se∼Uniform(0.5,1.0) 72.04 50.87–98.26 Sp∼Uniform(0.7,1.0) 98.74 97.27–99.99 Scenario4 TP∼Uniform(0,0.05) 1.95 0.01–4.50 Se∼Uniform(0.4,1.0) 66.11 41.0–97.7 Sp∼Uniform(0.6,1.0) 98.71 97.7–99.8 Scenario5 TP∼Uniform(0,1) 1.50 0.005–4.30 Se∼Beta(8.0,4.0) 63.63 34.63–87.83 Sp∼ Beta(9.0,2.0) 98.31 97.05–99.65 Scenario6 TP∼Uniform(0,1) 0.3 0.01–1.25 Se∼ Uniform(71,31) 69.28 60.01–78.03 Sp∼Uniform(86,16) 96.86 95.00–97.7 Scenario7 TP∼Uniform(0,1) 1.50 0.005–4.69 Se∼ Uniform(5.28,2.07) 67.02 68.8–94.6 Sp∼Uniform(21.20,2.06) 98.34 97.08–99.64

Theagreementamong thedifferentlaboratory-based

diagnosticmethodsappliedinthisstudywasassessedby

Cohen kappastatistic under cluster sampling(svykappa

function) under R environment, described by Lumley

(2004), which is the approach used to asses different

testswithoutassumingthatone isthebest.Theresults

obtainedwereclassiﬁedaccordingtotheAltmanscaleto

giveagradeofsigniﬁcance(i.e.,>0.80:verygood

agree-ment, 0.61–0.80:good agreement, 0.41–0.60:moderate

agreement, 0.21–0.40: fair agreement, and ≤0.20: poor

agreement)(Abraira,2000).Inaddition,theSeandSpof

histopathology,microscopy,andPCRwerecalculated

tak-ingtheinvitrocultureas agoldstandard test.The95%

conﬁdentintervals(CI)wereestimatedfor SeandSpof

eachtest.

3. Results

Theapparentprevalenceofgrossvisiblelesionsfound

byveterinary inspection was2.33% (95%CI=1.37–3.73)

and 2.42% (95% CI=1.41–3.84) for the 2007 and 2008

surveysrespectively.The TPwascalculated tobe1.50%

(95%PI=0.005–4.69)takingintoaccountourpriorbelieves

related with the characteristic of this test. The

esti-matedSe andSp ofnecropsyin this studywere67.02%

(95%PI=29.39–94.58)and98.34%(95%PI=97.08–99.64),

respectively.TheestimatesoftheTPdidnotshow

impor-tantdifferencesunderseveralconstrains,theywerearound

1.5%,althoughwhenconstrainshadlowuncertainty

(sce-nario6)theestimatesofTPwaslower.Theestimatesfor

Spofnecropsywereingeneralconsistentandhigh.The

estimatesforSeofnecropsyvariedaccordingtothe

con-strictionsandwideintervalswerefound(Table1).

Macroscopically, we observed yellowish

granuloma-toustuberclesinmediastinal (51.32%),tracheobronchial

(23.68%),hepatic (11.84%), and retropharyngeal (9.21%)

lymphnodes.Onlyoneanimalshowedvisiblelunglesions.

Also,theﬁveCITTreactorsshowedvisiblelesionsin

tho-raciclymphnodes,butnotinthelungs.

Fromthe94specimens,31yieldedoneormorepositive

resultsinthelaboratory(Table2):all24positivelymph

nodesfrom15cattleshowedvisiblelesions(8bronchial,7

mediastinal,3hepatic,3mammary,2retropharyngeal,and

1scapularlymphnode),versusonlyoneofthe7(14.28%)

positivelungtissues(Table2).Fourteenof31specimens

yieldedapositiveresultinonlyasingletest,6scored

pos-itivein2tests,6in3tests,and5hadapositiveresultinall

4testsapplied.

Ontheanimallevel,thisresultedinthedetectionofM.

bovisin51.5%(17/33)ofthesuspectedanimals.In7

ani-mals,onlyonespecimenwasfoundpositive,whereasthe

remaininganimalshad2–4positivespecimens.

Histopathologyexamination allowedforthe

identiﬁ-cation of BTBin 27.3% (9/33) ofthe suspectedanimals.

It showed focal necrosis, distrophic calciﬁcation and/or

Langhans-andforeignbody-typegiantcellsinatotalof

12biopsies.Giantcellswereidentiﬁedinthelung

sam-ple showing gross lesions and in 3 mediastinal lymph

nodes.Necrosisand/orcalciﬁcationweremorefrequently

observed(11samples).Intheremaininganimals(24/33),a

varietyoflesionswasidentiﬁedinlungs(chronic

intersti-tialpneumonia65.7%,atelectasis17.1%,emphysema8.6%,

granulomatous pneumonia 5.7% and bronchioloalveolar

carcinoma2.9%),andlymphnodes(caseous

lymphadeni-tis 38.3%, follicular hyperplasia 31.9%, lymphoma 8.5%,

oedemawithinﬁltrateofmacrophages6.3%,otherlesions

4.2%,andnosigniﬁcantlesions10.6%).

AFB weredetectedin ZN-stainedsmearsfrom 33.3%

(11/33)ofsuspectedanimals.Fourteen(14)of94smears

werepositive:4fromlung samplesand 10fromlymph

nodes,mainlybronchialandmediastinal(Table2).

How-ever,thenumberofAFBdetectedpersmearwasverylow,

withamaximumof10.Fivecaseshadmorethan3AFBper

slidebutin9slidesonly1or2AFBwereobserved.

Invitro cultureprovedthemostsensitivemethodin

thisstudy.M.boviswasisolatedfrom36.4%(12/33)

sus-pectedcattle.Bacilliweregrownin23samplesafterabout7

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Table2

Distributionofpositivelaboratoryresultsforbovinetuberculosisobservedin31specimensfrom17differentanimalstakenfromdairycattleatthe slaughterhouseofMachachi,Ecuador.

Area Animalidentiﬁcation Farm Specimen Visiblelesion SmearZN CultureST PCR16SrRNA Histo-pathology

Machachi 1 A BronchialLN Yes Positive – – –

5 B Lung No – – Positive –

8 C BronchialLN Yes – Positive – Positive

RetropharyngealLN Yes – – – Positive

33 C Lung No – Positive

14 C RetropharyngealLN Yes – Positive – Positive

BronchialLN Yes – Positive Positive Positive

HepaticLN Yes – Positive Positive –

MammaryLN Yes – Positive Positive –

Lung No Positive Positive Positive –

23 C Lung No Positive – – –

MediastinalLN Yes – Positive – –

13 D HepaticLN Yes – Positive Positive Positive

BronchialLN Yes – Positive – –

Lung No – – Positive –

26 D MediastinalLN Yes Positive Positive Positive Positive

30 D MediastinalLN Yes – Positive – –

32 D MediastinalLN Yes – – – Positive

Tambillo 12 E BronchialLN Yes Positive Positive – –

Lung No Positive Positive Positive Positive

25 F MammaryLN Yes Positive – – –

Aloasi 16 G ScapularLN Yes – Positive – –

BronchialLN Yes Positive Positive Positive –

MediastinalLN Yes – Positive – Positive

31 G MammaryLN Yes Positive – – –

28 H BronchialLN Yes Positive Positive Positive –

Aloag 19 I Lung Yes Positive Positive Positive Positive

MediastinalLN Yes Positive Positive Positive Positive

BronchialLN Yes Positive Positive Positive –

HepaticLN Yes – Positive – –

Chaupi 20 J MediastinalLN Yes Positive Positive Positive Positive

ZN=Ziehl–Neelsen,ST=stonebrink,LN=lymphnode.

obtainedfrom19lymphnodeand4lungspecimens,all

positiveM.bovisisolateswereconﬁrmedby

spoligotyp-ing.NomycobacteriaotherthanM.bovisweredetectedby

culture.

Nested PCR detected27.23% (9/33) positive animals.

M.tuberculosis complexDNA wasampliﬁedin15 of 94

samples:10 lymphnodesand 5lungbiopsies(Table2).

NegativeDNA-extractionandPCRcontrolsyieldeda

nega-tiveresultinallPCRruns.

Survey kappaanalysisof thevariouslaboratorytests

revealedasubstantialconcordancebetweencultureand

PCR (=0.61), a moderate concordance between

cul-tureandhistopathology(=0.49),cultureandmicroscopy

(=0.44), PCR and microscopy (=0.47), and PCR with

histopathology (=0.44) (Table 3). A low concordance

was observed between microscopy and histopathology

(=0.20).

Comparedtoinvitroculture,the3remainingtechniques

hadalowsensitivitytodetectBTB.TheSeofPCR(56.5%;

95% CI=34.4–76.8)was higher than thatof microscopy

(43.5%;95%CI=23.2–65.5)andhistopathology(43.5%;95%

CI=23.2–65.5).Speciﬁcitiesweremuchhigherwith94.4%

(95%CI=86.2–98.4) for PCR and microscopy,and 97.2%

(95%CI=90.2–99.6)forhistopathology.

4. Discussion

The prevalenceof BTB, asdetermined by the

detec-tionofgrossvisible lesionsinslaughteredanimals,was

similarin2007and2008(2.32%and2.41%,respectively).

However,thetrueprevalence,takingintoaccounttheSe

(83.33%)(Norbyetal.,2004)and Sp(95%)calculatedfor

thismethod,waslower(1.50%).Asthisstudywastheﬁrst

ofitskindintheMejiacanton,we cannotcompareour

Table3

ConcordanceanalysisbysurveysamplingKappaofthelaboratorytestsappliedtodiagnoseBTBfromspecimenstakenattheabattoiroftheMejia canton—Ecuadorduring2007and2008.

Tests Microscopy Culture PCR Histopathology

value Stderror value Stderror value Stderror value Stderror

Microscopy 1 na

Culture 0.44 0.12 1 na

PCR 0.47 0.15 0.61 0.08 1 na

Histopathology 0.20 0.14 0.48 0.06 0.44 0.13 1 na

(6)

resultswithpreviousdataofthis region,eventhoughit

isthemostimportantdairyregioninthenorthofEcuador.

Abattoirsurveysperformedinotherprovincesshoweda

lowerprevalence:0.12%and0.46%inGuayas,and0.21%

inSantoDomingodelosSáchilas(Mata,1973;Cuetoand

Suárez, 1993; Coloma, 2000).However, thesestudies –

whichwereneverofﬁciallypublished–werecarriedout

inmeatproductionregionsandthereforearenot

compa-rablewithourdatafromdairycattleasshownbydatafrom

othercountries.InMexico,anabattoirsurveyrevealedthe

presenceofgrosslesionstypicalforBTBin16%ofdairy

cattle(Milian-Suazoet al., 2000), whereas another

sur-veyrevealed signiﬁcantlyless grosslesionsamong beef

cattle(0.05%)(BrownanddeAnda,1998).Thisdifference

betweendairyandbeefcattlehasbeenobservedin

differ-entabattoirsthroughoutEcuador,anditwasattributedto

severalfactorssuchascattledensity,management

prac-ticesandbreed;however,datahaveneverbeenpublished

(personalcommunication).InEcuadoriandairyindustry,

importedEuropeanbreedshavebeenusedtoimprovemilk

production,i.e. HolsteinFriesian,which is less resistant

toBTBthanZebu(Omeretal.,2001;Amenietal.,2007; Kazwalaetal.,2001).

ComparedtotherecentlyobservedCITT-based

preva-lence data among dairy cattle from the Mejia canton

(Proa ˜no-Pérez et al., 2009), our

veterinary-inspection-basedprevalencewaslower.Thiscouldreﬂectthelower

sensitivity of post-mortem examination (Norby et al.,

2004), implicatingthatahighnumberofanimalsmight

beclassiﬁed as negativeby this method.Routine

abat-toirinspectioncanbeaffectedbythemethodemployed,

theanatomicalsitesexaminedandthestageofinfection

(Corner, 1994);it willfail to detectinfected animals if

lesionsarepresentintissuesnotbeinginspected.Sincethe

veterinaryinspectioninoursurveywasdoneinavery

com-prehensiveway,webelievethattheearlystageofinfection

playedaroleinthelowdetectionrate.Duringour

abat-toirsurvey only one of the5 bovine CITT reactors had

evidentgrosslesions;therestshowedonlyminorlesions

inthoraciclymphnodes.Threeofthelatterwere

identi-ﬁedasCITT-reactorsintheyearofslaughter,andtherefore

mightbeattheearlystageofinfection.Thetworemaining

animalswereCITT-reactorsintwoconsecutiveyearsand

slaughteredafterwards;oneofthemshowed

characteris-ticsofadvanceddisease.Nevertheless,BTBwasconﬁrmed

bylaboratorytestsinallCITT-reactors.Theseﬁndings

con-ﬁrmthatanimalsonlyshowevidentcharacteristicsinan

advancedstageofthedisease(Corner,1994),althoughthe

symptomsofthediseasearenotpathognomonic.Onthe

otherhand,ourdataalsoconﬁrmthatM.boviscanbe

iso-latedfromanimalswithoutgrosslesions,includinglungs

(Whippleetal.,1996;Tekluletal.,2004).

Grossvisiblelesionsmaybecausedbyotherorganisms

aswell,andalsoNTMmayconstituteaconfoundingfactor.

InTanzania,Cleavelandandcolleagues(2007)identiﬁedM.

terrae,M.avium,M.chelonae,M.gordonae,M.fortuitum,M.

ﬂavescensandM.smegmatisinsamplesfromslaughtered

cattlewithvisiblelesions;however,theclinicalrelevance

oftheisolationoftheseenvironmentalmycobacterianeeds

tobeconﬁrmed.In Ecuador, thepresence ofM.

avium-intracellulare-scrofulaceum,M.gordonae,M.szulgaiandM.

celatumwasreportedinslaughtereddairycattle(Proa ˜

no-Perez et al., 2006). Therefore, the identiﬁcation of the

species playsa crucial role in theﬁnal diagnosisofthe

disease.WedidnotisolateNTMinthepresentstudy.

The combined use of microscopy, in vitro culture,

PCR and histopathology identiﬁedBTB in 51.5% (17/33)

of the suspectedanimals. Nevertheless, only6 of these

animals were positive by all the laboratory methods

used.

M.boviswasidentiﬁedbyinvitroculturein36.34%of

thesuspectedcattle.Thisislowcomparedtostudiesfrom

MexicoandArgentina.InMexico,BTBinfectionwas

con-ﬁrmedbyculturein59%ofcattlefrom6importantdairy

regions(Milian-Suazoetal.,2000),andintheProvinceof

SantaFé,Argentina,with83%ofsamplesshowingvisible

lesions (Latini et al., 1997).The Seof detection by

cul-turecanbereducedduetotheapplieddecontamination

procedureorpre-analyticalstorageconditionsofthe

sam-ples,affectingtheviabilityoftheMycobacteria(Palomino

and Portaels,1998). Freezing–thawing cyclesproved to

reducetheviabilityofmycobacteriasigniﬁcantly(Portaels

etal.,1988).Asourspecimensunderwenttwofreeze–thaw

cycles,partofthebacillimayhavebeenkilledand

there-forecouldnotbeisolated.AFBweredetectedin14of94

specimens,butin4ofthemthepresenceofM.boviscould

notbeconﬁrmedbyculture.Thismightbeattributedtothe

suboptimalstorageconditionsnotensuringsurvivalofthe

bacteria.Nevertheless,ourculturepositivityratewasstill

higherthanthatobservedinsomeotherstudies.InBrazil,

culturingofM.boviswassuccessfulin18%ofthesamples

(PiresdeAraújoetal.,2005);noinformationwasgivenon

thestorageconditionsoftheinvestigatedsamples.

ThedetectionratebyPCRwasevenlowerinourstudy.

Appliedondecontaminatedtissuesamples,itcoulddetect

BTBin27.3%ofsuspectedanimals.In Brazil,18%of

cat-tleshowinggrosslesionsyieldedapositivePCR(Piresde

Araújo etal.,2005).TheSeof nucleicacidampliﬁcation

canbeinﬂuencedbydifferentfactors,suchasinsufﬁcient

quantityofbacilliorthepresenceofinhibitorshampering

thepolymerase(Mangiapanetal.,1996;PiresdeAraújo

et al.,2005;Cardoso etal., 2009).In Brazil,comparison

between different concentrations and volumes of DNA

samplesshowedthatthepercentageofPCR-positivelymph

nodesincreasedfrom39.4%to54.5%afterre-analysisof

theinitiallynegativesamplesusinga1:2dilutionofthe

suspensionand2,5␮linsteadof1␮l(Cardosoetal.,2009).

Inourstudy,weused10␮lofundilutedDNAextractfor

thePCR,implicatingthatthesensitivityofourPCRmight

increasebytheuseofdilutedDNAextracts,thus

minimiz-ingtheeffectofpossibleinhibitors.

We observedagoodagreementbetweencultureand

PCR(=0.61),implicatingthatahighnumberofsamples

yieldedapositiveresultbybothtests,andconﬁrmingthat

cultureandPCRarethemostimportantdiagnostictoolsfor

thediagnosisofBTBindairyareas(Cardosoetal.,2009).

HistopathologyshowedBTBcompatibleimagesin27.3%

of the cattle with macroscopiclesions. Demelash et al.

(2009)showedthattheSeofhistopathologydiagnosiscan

beaffectedbythetechniqueusedandthenumberofslides

examined. In our study, two slides per specimen were

(7)

hand,only5ofthe9animalsidentiﬁedbyhistopathology

werepositivebyalllaboratorytests.

Thediagnosticaccuracyofatestisprimarilydeﬁnedby

itsSeandSp.However,theseparameterscanbeinﬂuenced

by minor changes occurring when processing samples.

In this study,culture wasconsideredthegoldstandard

methodtoestimatetheSeandSpoftheremaining

diag-nostic tests.It isimportant toconsider thatallsamples

werepaucibacillaryandpre-analyticalsampleconditions

weresuboptimalforculturebecauseofthetwo

freezing-thawingcycles.Therefore,theobservedsensitivitiesofPCR

(56.5%),microscopyandhistopathology(43.5%each)could

beoverestimated.MicroscopyisknowntohavealowSe,

asitrequires5000to10,000bacilli/mLofsampletoyielda

positiveresult.TheSeofPCRandhistopathologycouldbe

inﬂuencedbyfactorsdescribedabove.

Inroutineconditions,itisnotfeasibletoapplyallthese

laboratorytestsforallsamples,giventhehighcost and

labourrequired.Therefore,thebeststrategyshouldbe

cho-sentoconﬁrmthesuspectedcaseswhenimplementinga

BTBcontrolprograminEcuador.Theresultsobtainedin

thisstudysuggestthatPCRisagoodalternativefor

cul-ture,yielding resultsin a much shorter time. However,

as it counts for culture,PCR needs tobeperformed by

trainedpersonalinanappropriate,quality-ensured

labo-ratory.Ontheotherhand,microscopyisrapid,easy,and

cheap,anditcanbeimplementedinresource-poorsettings

(Hiraoetal.,2007).Becauseofitslowsensitivity,all

smear-negativesamplesshouldberetestedbycultureand/orPCR.

Apossiblestrategytoperformpost-mortemdiagnosisfor

BTBcouldbeasfollows:veterinaryinspection,sampling

oftissuesshowinggrossvisiblelesionsandlungs,smear

microscopytodetectAFB,andPCRonallsmear-negative

samples.

Our second objective was tostudy and characterize

the distributionof lesions observedin the organs from

slaughteredcattleasasurrogatemarkerforthepossible

routeoftransmissionandstageofdisease(Phillipsetal.,

2003).Duringoursurvey,macroscopiclesionswerefound

inmediastinal(51.32%),tracheobronchial(23.68%),hepatic

(11.84%)andretropharyngeallymphnodes(9.21%),andin

othersites(3.95%).Mildlesionsinlungswerefoundinonly

one slaughteredbovine.Similarobservations were

pub-lishedintheUnitedStatesandIran,wherenogrosslesions

suggestiveofBTBwereobservedinlungsfromdairy

cat-tle,butfrequentlyinlymphnodesofthethoracicregion

(Whippleetal.,1996;Tadayonetal.,2008).Onthe

con-trary,affectionoflungsandotherorganshasbeenreported

incattleinAfrica.InEthiopia,84%ofvisiblelesionswere

foundinthelungsandthoraciclymphnodes(Tekluletal.,

2004).InMali,79%oftheanimalshadconﬁrmedlesions

inlungs,andtheinfectionwithM.boviswashighly

asso-ciatedwiththeselesions(p<0.001)(Mülleretal.,2008).

However,inTanzania,visiblelesionsweremainlyfoundin

thegastrointestinaltract(61.3%)(Cleavelandetal.,2007);

thesedifferencescouldbeattributedtothebreedsinvolved

inthestudies.

ItisimportanttoalsoconsiderthatdifferentM.bovis

genotypesmaydifferinpathogenesisandtherebyaffect

thedevelopmentoflesions(Goodchildetal.,2003;Ameni

et al., 2007; Oloya et al., 2007). In our study all

ani-malswithmacroscopiclesionswereHolsteinFriesianand

infectedbythesamestrain(SB0980;Proa ˜no-Pérezetal.,in

preparation).Theobservedinter-animalheterogeneityof

macroscopicandmicroscopiclesionsthereforemostlikely

representstheindividualdiversityofpathological

evolu-tionofBTB(Meikleetal.,2007).

Themacroscopiclesionsfoundindairycattle

slaugh-teredduringthissurveysuggestthatthemostimportant

routeoftransmissionamongthecattlestudiedwasthrough

respiratorytract.

5. Conclusion

In conclusion, our data demonstrate that veterinary

inspectionincombinationwithlaboratory-basedanalyses

isusefultodocumentthepresenceofBTB,andtheyurge

fortheimplementationofanationalBTBcontrolprogram.

Itshouldcombineinvivoskintestingbeforeslaughterto

identifyearlyinfectionswithM.bovis,withextended

post-morteminspectionincludinglaboratory-baseddiagnosis.

Theseactivitiesshouldbeperformedbyqualiﬁed

veteri-nariansandtechnicians,followingstrictsafetyprocedures

toavoidhumaninfections.Veterinaryinspectionneedsto

beimplemented as a routine procedurein all abattoirs

locatedinareaswithahighcattledensity.However,this

canonlybeachievedifBTBisdeclaredanimportantdisease

foranimalandhumanhealthinEcuador,andtherequired

ﬁnancialsupportisprovidedtosupportinspections,testing

andcompensationforlosses.

Acknowledgements

Theauthorsgratefullyacknowledgetheﬁnancial

sup-portfrom‘CommissionUniversitairepourle

Développe-ment’ (CUD), Universityof Liege– Project PIC. We also

thankDr.AntonioViteriandallslaughterhouseworkers

intheMejiacantonfortheircollaborationduringour

sur-vey.Finally,wethanktheimportantcontributionofthe

ReviewersandAssociateEditorfromthisJournal.

References

Abraira,V.,2000.ElindiceKappa.Semergen27,247–249.

Ameni,G.,Aseffa,A.,Engers,H.,Young,D.,Gordon,S.,Hewinson,G., Vorde-meier,M.,2007.Highprevalenceandincreasedseverityofpathology ofbovinetuberculosisinHolsteinscomparedtozebubreedsunder ﬁeldcattlehusbandryinCentralEthiopia.Clin.Vacc.Immunol.14, 1356–1361.

Berkvens,D.,Speybroeck,N.,Praet,N.,Adel,A.,Lesaffre,D.,2006. Esti-matingdiseaseprevalenceinBayesianframeworkusingprobabilistic constraints.Epidemiology17,145–153.

Branscum,A.J.,Gardner,I.A.,Johnson,W.O.,2005.Estimationofdiagnostic testsentitivityandspeciﬁcitythroughBayesianmodelling.Prev.Vet. Med.68,145–163.

Branscum,A.J.,Gardner,I.A.,Johnson,W.O.,2004.Bayesianmodellingof animalandherdlevelprevalences.Prev.Vet.Med.66,101–112. Brown,W.H.,deAnda,J.H.,1998.Tuberculosisinadultbeefcattleof

Mex-icanoriginshippeddirect-to-slaughterintoTexas.J.Am.Vet.Med. Assoc.212,557–559.

Cardoso,M.A.,Cardoso,R.F.,Hirata,R.D.C.,Hirata,M.H.,Leite,C.Q.F., San-tos,A.C.B.,Siqueira,V.L.D.,Okano,W.,Rocha,N.S.,Lonardoni,M.V.C., 2009.DirectdetectionofMycobacteriumbovisinbovinelymphnodes byPCR.ZoonosesPublicHealth56,465–470.

Cleaveland,S.,Shaw,D.J.,Mﬁnanga,S.G.,Shirima,G.,Kazwala,R.R.,Eblate, E.,Sharp,M.,2007.MycobacteriumbovisinruralTanzania:riskfactors forinfectioninhumanandcattlepopulations.Tuberculosis87,30–43.

(8)

Collins,J.D.,2006.Tuberculosisincattle:strategicplanningforthefuture. Vet.Microbiol.112,369–381.

Coloma,L.,2000.Determinacióndetuberculosisenbovinossacriﬁcados enelmataderomunicipaldeSantoDomingodelosColorados[Thesis endofstudies].Guayaquil:FacultyofVeterinaryMedicine,Agraria UniversityofEcuador.

Corner,L.A.,1994.PostmortemdiagnosisofMycobacteriumbovisinfection incattle.Vet.Microbiol.40,53–63.

Cosivi,O.,Grange,J.M.,Daborn,C.J.,Raviglione,M.C.,Fujikura,T.,Cousins, D.,Robinson,R.A.,Huchzermeyer,H.F.,deKantor,I.,Meslin,F.X.,1998. ZoonotictuberculosisduetoMycobacteriumbovisindeveloping coun-tries.Emerg.Infect.Dis.4,59–70.

Cueto,N.,Suárez,M.,1993.Porcentajedelesionestuberculosas encon-tradasenanimalesdeabastoaniveldemataderoysuveriﬁcacióncon diagnosticobacteriológico[Thesisendofstudies].Guayaquil:Faculty ofVeterinaryMedicine,AgrariaUniversityofEcuador.

Demelash,B.,Inangolet,F.,Oloya,J.,Asseged,B.,Badaso,M.,Yilkal,A., Skjerve,E., 2009. Prevalenceofbovinetuberculosis inEthiopian slaughtercattlebasedonpost-mortemexamination.Trop.Anim. HealthProd.41,755–765.

Enoe,C.,Georgiadis,M.P.,Johson,W.O.,2000.Estimationofsensitivity andspeciﬁcityofdiagnostictestanddiseaseprevalencewhenthe truediseasesstateisunknown.Pre.Vet.Med.45,61–81.

Goodchild,A.V.,DelaRua-Domenech,R.,Palmer,S.,Dale,J.,Gordon, S.V.,Hewinson,R.G.,Clifton-Hardley,R.S.,2003.Associationbetween moleculartypeandepidemiologicalfeaturesofMycobacteriumbovis incattle.In:TheSocietyforVeterinaryEpidemiologyandPreventive Medicine,UniversityofWarwick,pp.45–59.

Goodchild,A.V.,Clifton-Hadley,R.S.,2001.Cattle-to-cattletransmission ofMycobacteriumbovis.Tuberculosis(Edinb).81,23–41.

Hirao,S.,Yassin,M.A.,Khamofu,H.G.,Lawson,L.,Cambanis,A.,Ramsay,A., Cuevas,L.E.,2007.Same-daysmearsinthediagnosisoftuberculosis. Trop.Med.Int.Health.12,1459–1463.

Kamerbeek,J.,Schouls,L.,Kolk,A.,VanAgterveld,M.,VanSoolingen,D., Kuijper,S.,Bunschoten,A.,Molhuizen,H.,Shaw,R.,Goyal,M.,Van Embden,J.,1997.Simultaneousdetectionandstraindifferentiation ofMycobacteriumtuberculosisfordiagnosisandepidemiology.J.Clin. Microbiol.35,907–914.

Kazwala,R.,Kambarage,D.,Daborn,C.,Nvange,J.,Jiwa,S.,Sharp,J.,2001. Riskfactorsassociatedwiththeoccurrenceofbovinetuberculosisin cattleintheSouthernHighlandsofTanzania.Vet.Res.Commun.25, 609–614.

Latini,O.,Canal,A.M.,Ferrara,M.E.,Sequeira,M.D.,Sequeira,G.,Bagnaroli, R.,Torres,P.,1997.Conﬁabilidadenladeterminacióndeprevalencia deinfecciónporMycobacteriumbovisenganadobovinopordecomisos enfrigoríﬁcos.Arch.deMed.Vet.29,197–204.

Lumley,T.,2004.Analysisofcomplexsurveysamples.J.Stat.Softw.9,8. Mangiapan,G.,Vokurka,M.,Schouls,L.,Cadranel,J.,Lecossier,D.,van

Embden,J.,Hance,A.J.,1996.Sequencecapture-PCRimproves detec-tionofmycobacterialDNAinclinicalspecimens.J.Clin.Microbiol.34, 1209–1215.

Mata,M.,1973.Determinacióndelíndicedebovinostuberculososquese sacriﬁcanenelmataderomunicipaldelacuidaddeGuayaquil[Thesis endofstudies].Guayaquil:FacultyofVeterinaryMedicine,University ofGuayaquil.

Meikle,V.,Schneider,M.,Azenzo,G.,Zumárraga,M.,Magnano,G.,Cataldi, A.,2007.Individualanimalsofacattleherdinfectedwiththesame Mycobacteriumbovisgenotypeshowsimportantvariationsin bac-teriological,histopathological and immune response parameters. ZoonosesPublicHealth54,86–93.

Milian-Suazo,F.,Salman,M.D.,Ramirez,C.,Payeur,J.B.,Rhyan,J.C., San-tillan,M.,2000.Identiﬁcationoftuberculosisincattleslaughteredin Mexico.Am.J.Vet.Res.61,86–89.

Müller,B.,Steiner,B.,Bonfoh,B.,Fané,A.,Smith,N.H.,Zinsstag,J.,2008. MolecularcharacterisationofMycobacteriumbovisisolatedfrom cat-tleslaughteredattheBamakoabattoirinMali.BMCVet.Res.4, 26.

Neill, S.D., Pollock, J.M., Bryson, D.B., Hanna, J., 1994. Pathogene-sisofMycobacterium bovisinfectionin cattle.Vet.Microbiol.40, 41–52.

Norby,B.,Bartlett,P.C.,Fitzgerald,S.D.,Granger,L.M.,Bruning-Fann,C.S., Whipple,D.L.,Payeur,J.B.,2004.Thesensitivityofgrossnecropsy, cau-dalfoldandcomparativecervicaltestsforthediagnosisofbovine tuberculosis.J.Vet.Diagn.Invest.16,126–131.

Oloya,J.,Kazwala,R.,Lund,A.,Opuda-Asibo,J.,Demelash,B.,Skjerve, E.,Johansen,T.B.,Djønne,B.,2007.Characterisationof mycobacte-riaisolatedfromslaughtercattleinpastoralregionsofUganda.BMC Microbiol.7,95.

Omer,M.K.,Skjerve,E.,Woldehiwet,Z.,Holstad,G.,2001.Across-sectional studyofbovinetuberculosisindairyfarmsinAsmara,Eritrea.Trop. Anim.HealthProd.33,295–303.

Palomino,J.C.,Portaels,F.,1998.Effectsofdecontaminationmethods andcultureconditionsonviabilityofMycobacteriumulceransinthe BACTECSystem.J.Clin.Microbiol.36,402–408.

Parks,L.C.,1997.MicrobiologicalMedia,Secondedition.CRCPress,New York,pp.774–776.

Phillips,C.J.,Foster,C.R.,Morris,P.A.,Teverson,R.,2003.Thetransmission ofMycobacteriumbovisinfectiontocattle.Res.Vet.Sci.74,1–15. PiresdeAraújo,C.,Leite,C.Q.,AndradedePrince,K.A.,Jorge,K.,Osório,A.L.,

2005.Mycobacteriumbovisidentiﬁcationbyamolecularmethodfrom post-morteminspectedcattleobtainedinabattoirsofMatoGrossodo Sul,Brazil.Mem.Inst.OswaldoCruz.100,749–752.

Pollock,J.M.,Rodgers,J.D.,Welsh,M.D.,McNair,J.,2006.Pathogenesisof bovinetuberculosis:theroleofexperimentalmodelsofinfection.Vet. Microbiol.112,141–150.

Portaels, F.,Realini, L., Bauwens, L., Hirschel, B., Meyers, W.M., de Meurichy, W., 1996. Mycobacteriosis caused by Mycobacterium genavenseinbirdskeptinazoo:11-yearsurvey.J.Clin.Microbiol. 34,319–323.

Portaels,F.,Fissette,K., DeRidder,K.,Macedo,P.M.,DeMuynck, A., Silva,M.T.,1988.Effectsoffreezingandthawingontheviabilityand theultrastructureofinvivogrownmycobacteria.Int.J.Lepr.Other Mycobact.Dis.56,580–587.

Proa ˜no-Pérez, F., Benítez-Ortiz, W., Celi-Erazo, M., Ron-Garrido, L., Benítez-Capistros,R.,Portaels,F.,Rigouts,L.,Linden,A.,2009. Com-parativeintradermaltuberculintestindairycattleinthenorthof Ecuadorandriskfactorsassociatedwithbovinetuberculosis.Am.J. Trop.Med.Hyg.81,1103–1109.

Proa ˜no-Perez,F.,Rigouts,L.,Brandt,J.,Dorny,P., Ron,J.,Chavez,M., Rodriguez,R.,Fissette,K.,VanAerde,A.,Portaels,F.,Benitez-Ortiz,W., 2006.PreliminaryobservationsonMycobacteriumspp.indairycattle inEcuador.Am.J.Trop.Med.Hyg.75,318–323.

Spiegelhalter, D., Thomas, A., Best, N., Gilks, W., 1996. BUGS: Bayesian Inference Using Gibbs Sampling, Version 0.50. MRC Biostatistics Unit, Cambridge. http://www.mrc-bsu.cam.ac.uk/ bugs/winbugs/contents.shtml.

Tadayon,K.,Mosavari,N.,Sadeghi,F.,Forbes,K.J.,2008.Mycobacterium bovisinfectioninHolsteinFriesiancattle,Iran.Emerg.Infect.Dis.14, 1919–1921.

Teklul,A.,Asseged,B.,Yimer,E.,Gebeyehu,M.,Woldesenbet,Z.,2004. Tuberculouslesionsnotdetectedbyroutineabattoirinspection:the experienceoftheHossanamunicipalabattoir,southernEthiopia.Rev. Sci.Tech.23,957–964.

Whipple,D.L.,Bolin,C.A.,Miller,J.M.,1996.Distributionoflesionsin cattleinfectedwithMycobacterium bovis.J. Vet.Diagn.Invest.8, 351–354.