Malignant Catarrhal Fever in Brazilian cattle presenting with neurological syndrome

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Malignant

Catarrhal

Fever

in

Brazilian

cattle

presenting

with

neurological

syndrome

Maira

de

S.N.

Martins

_,

_Alessandra

_M.M.G.

_de

_Castro

_,

_Michele

_dos

_S.

_Lima

_,

Vivian

da

S.C.

Pinto

_,

_Thaís

_G.

_da

_Silva

_,

_Claudia

_Del

_Fava

_,

_Claudio

_Regis

_Depes

_,

Liria

H.

Okuda

_,

_Edviges

_M.

_Pituco

a_{InstitutoBiológico,LaboratóriodeVirosesdeBovídeos,SãoPaulo,SP,Brazil} b_{ComplexoEducacionalFaculdadeMetropolitanasUnidas,SãoPaulo,SP,Brazil} c_{CoordenadoriadeDefesaAgropecuáriadoEstadodeSãoPaulo,SãoPaulo,SP,Brazil}

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Articlehistory:

Received8March2016 Accepted17October2016 Availableonline2January2017 AssociateEditor:MilianeSouza

Keywords:

Ovineherpesvirustype2(OvHV-2) Histopathology

QualitativePCR QuantitativePCR Phylogeneticanalysis

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MalignantCatarrhalFever(MCF)wasinvestigatedinthecentralnervoussystemofcattle withneurologicalsyndrome.Two-hundred-ninetysampleswereanalyzedbyhistology,and molecularmethodstodetectovineherpesvirustype2(OvHV-2)wereoptimizedand val-idated.Thequalitativepolymerasechainreaction(qualitativePCR)analyticalsensitivity was101_{DNAcopies/␮Landfound4.8%(14/290)positiveforOvHV-2.Thequantitative}

poly-merasechainreaction(qPCR)analyticalsensitivitywas100_{DNAcopy/␮Land5.9%(17/290)}

positivity,with47.1%(8/17)ofthepositivesamplespresentinghistologicalevidenceof non-purulentmeningo-encephalitis.ThequalitativePCRproducts(422bpoftheORF75region) weresequencedandsubmittedtophylogeneticanalysis.Identitymatricesshowed100% similarityinOvHV-2samplesobtainedinthisstudyandthoserecoveredfromGenBank, corroboratingotherstudies.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Encephalitisand encephalopathies have serious impacton publichealth,produceeconomiclosses,andareabarrierto internationaltrade.1_{Thedifferentialdiagnosisofbovine}

neu-rologicalsyndromeisessential,andischallengingduetothe difficultyinidentifyingpotentialagentsand obtaining con-clusiveresults. Diagnosisbeginswithinvestigations forthe mostprobableagents, and,inthe caseofnegativeresults, proceedingtoincludeotherpathogens.1

∗ _{Correspondingauthor.}

E-mail:mairamartins.bio@gmail.com(M.S.Martins).

MalignantCatarrhalFever(MCF)isawidespreaddisease that affects multiple systems and is often fatal to many artiodactyl species. Itis caused byHerpesvirus of the fam-ilyGammaherpesvirinaeandMacavirus,agroupofantigenic and genetically related viruses, known as MCF viruses,2

which cause clinical or subclinical infection insusceptible animals.3–5Todate,tenMCFviruseshavebeenidentified,with themostfrequentbeingovineherpesvirustype2(OvHV-2), whichcausesMCFinsheep(SA-MCF).6

DiagnosisofSA-MCFisgenerallymadefromclinical his-toryandsigns,non-specificlesionsobservedduringnecropsy,

http://dx.doi.org/10.1016/j.bjm.2016.10.021

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

histology,and epidemiologic data.7 _{Molecular methods are}

recommended,astheyallowviralDNAdetectionboth ante-andpost-mortem,toidentifythediseaseandassessits epi-demiology.

Lack of awareness of MCF as a cause of neurological syndrome in cattle and the importance of the differential diagnosisofneurologicaldiseasesreinforcestheneedfor opti-mizationand validationofspecificandsensitivediagnostic methodsforthemolecularidentificationoftheOvHV-2.

ThisresearchaimedtoevaluatesuspectedcasesofMCFin cattlebyclinicalandanatomopathologicalevaluationandby molecularcharacterizationinBrazil.

Materials

and

methods

Surveydesign

Retrospectivemolecularandhistologicalanalysesweremade of290centralnervoussystem(CNS)samplesfromcattlewith neurologicalsymptoms referred tothe Centro de Pesquisa e DesenvolvimentodeSanidadeAnimaldoInstitutoBiológicofor dif-ferentialdiagnosis ofneurological syndrome. The samples camefromseveralregionsofBrazil,with285collectedfrom January2012toMarch2014,onein2007,and four in2009. Allsampleswerenegativeforrabiesvirus,bovineherpesvirus

types1and5,bovineviraldiarrhea,andNeosporacaninum.

Standardvirusselection

A sample of bovine CNS from Assis County, São Paulo State was used as virus standard. The sample was found MCF-positive on histological examination. The viral DNA wasextractedusingTRizolaccordingtothemanufacturer’s instructions.AmplificationoftheORF75segmentthatencodes the phosphoribosylformylglycinamidine synthase (FGARAT) enzyme,participatinginpurinemetabolismandproduction ofviraltegumentproteins,wasconductedbyPCR,usingthe primersMCF556andMCF755,toamplifya422basepair(bp) segment.ThepositivePCRproductswerepurified,sequenced, andidentified asovineherpesvirus-type2(OvHV-2).Itwas quantifiedusingtheQuantiFluordsDNASystemfollowingthe

manufacturer’s instructions.ThepurifiedDNAwasusedas standardtooptimizeandvalidatemolecularmethods.

To determine the sensitivityof the molecular methods, ten-fold serial dilutions of a positive sample (100_–107_DNA

copies/␮L)wereperformedinnucleasefreewaterandinCNS negativeforOvHV-2.

Extractionofnucleicacid

TheDNAsampleswereextractedusingTRizol,accordingto the manufacturer’s instructions. The viral load was deter-minedbycomparingtothestandardcurve,expressedasDNA copiespergramtissue.

Optimizingandvalidatingmolecularmethods

PrimersandanhydrolysisprobetargetingtheORF75regions ofthe OvHV-2 genome were used. For qualitativePCR, the primersusedwereMCF556(5 GTCTGGGGTATATGAATC CAGATG GCTCTC 3),MCF755(5 AAGATAAGCACCAGT TATGCATCTGATAAA3),andMCF555(5TTCTGGGGTAGT GGC GAGCGAAGGCTTC 3).8 _{For qPCR,theprimersused}

wereforwardoF-OvHV-2(5TGGTAGGAGCAGGCTACCGT 3),reverseoR-OvHV-2(5ATCATGCTGACCCCTTGCAG3), andthehydrolysisprobeFAM/TAMRAoP-OvHV-2(5TCCACG CCGTCCGCACTGTAAGA3).9

ThetemperaturegradientwastestedinthequalitativePCR withMCF556vs.MCF755,MCF556vs.MCF555,andMCF755 vs.MCF555withvariousreagentconcentrationsandcycling conditions(datanotshown).

Reactionconditionsinbothamplificationswereadjusted to 12.5␮LPCRMaster Mix (Promega).Theconcentrationof externaland internal primerswas 0.25␮M (556and 755in thefirstamplified422bp,556and555inthesecond238bp). ThevolumeofDNAinthefirstandsecondamplificationwas 2.5␮Land2␮L,respectively,plusnucleasefreewatertoatotal volumeof25␮L.

QuantitativePCRusedSYBRGreenandTaqMansystemsat primerconcentrationsof200–1000nM.Thehydrolysisprobe wasat60–100nMconcentration(datanotshown).All reac-tionswerecarriedoutinaLightCycler480(Roche).

Table1–CyclingandtemperatureconditionsofthefirstandsecondamplificationforqualitativePCRandbothqPCR systems(SYBRGreenandTaqManSystems)forOvHV-2.

Method System Primers Initial denaturing 30cycles Final extension Curveof melting DNA denaturing Primer hybridization Polymerization Qualitative PCR First amplification: MCF556/755 95◦C/3min 95◦C/30s 60◦C/30s 72◦C/30s 72◦C/3min Second amplification: MCF556/555 95◦_{C/3min 95}◦_{C/30s 67}◦_{C/30s 72}◦_{C/30s 72}◦_C/3min Quantitative PCR SYBR

Green oF-OvHV-2 95◦C/5min

40cycles 72◦C5s/65◦C

1min/97◦_C

continuous 95◦C/10s 60◦C/20s 72◦C/6s

FortheSYBRGreenassay,acommercialkitLightCycler480 SYBRGreenIMaster(RocheMolecularSystems)wasusedwith 10␮LMasterMix,2×concentration.Theconcentrationofboth primerswas500nM; 2.0␮LDNA,and PCR-gradewatertoa totalvolumeof20␮L.

TheTaqMansystemwasimplementedusingthe LightCy-cler480ProbeMaster(RocheMolecularSystems)with10␮L Master Mix, 2× concentration. The concentration of both primerswas500nMandtheprobewas90nM;2.0␮LDNAand PCR-gradewatertoatotalvolumeof20␮L.

Followingtheoptimizedamplificationconditions(Table1), thestandardvirusdilutioninnuclease-freewater,aswellas inextractedDNAofCNSnegativeforOvHV-2,weresubmitted tothreereplicationsinthreedays,forvalidationand confir-mationofresultsoftheassay.10

Sequencing

A422bpfragmentfrom13positivesampleswassubmittedto sequencingusingtheAppliedBiosystemsABI3500XLGenetic Analyser(AppliedBiosystems).Thequalityofthesequences

wasassessedusingSequenceAnalyzer (AppliedBiosystem) software. BioEdit v. 7.1.11,11 _{BLAST and ClustalW software}

wereusedtoanalyzethesequences.Alcelophine Herpesvirus-1 was used as outgroup. Molecular Evolutionary Genetics Analysis(MEGA)v.6.012wasusedforphylogeneticanalysis. Thetreewasgeneratedbyaneighbor-joiningalgorithmusing Kimura2evolutionarymodelparameterswith1000bootstrap replicates.

Histology

Thesampleswereconventionallyprocessedandstainedwith hematoxylinandeosin13_{forhistologicalexamination.}

Results

Standardvirus

Theselectedstandardsamplewastheidentified798/07, col-lectedinAssisCounty,SãoPauloState,withMCFclinicaland histologicaldiagnosis(Fig.3).The422bpsequencepresented

Amplification curves Negative control 105 104 103 102 101 100 Select Zoom Select Zoom 34 543 31 543 28 543 25 543 22 543 19 543 16 543 13 543 10 543 7 543 4 543 1 543 12 477 11 477 10 477 9 477 8 477 7 477 6 477 5 477 4 477 3 477 2 477 1 477 0 477 65 70 75 80 85 90 95 5 35 Error: 108 Efficiency: 2 001 Slope: –3 319 Ylntercept: 35.63 30 25 20 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 10 15 Cycles Standard curve Log concentration Fluorescence (465–510) Crossing point 20 25 30 35 40 45 Melting peaks Temperature (ºC) -(d/dT) fluorescence (465–510)

C

B

A

Fig.1–StandardcurveSYBRGreensystem,dilutionsinextractsofCNSofcattlenegativetoOvHV-2.(A)Serialdilutions 100_–105_{DNAcopies/␮L.(B)Specificityofthereaction:efficacy=2.001,slope=−3.319.(C)Meltingcurvegeneratedinthe} SYBRGreensystem,dilutionsinextractsofCNSofcattlenegativetoOvHV-2withpeakatTm86◦C.

97–100%similaritytoOvHV-2fromseveralregionsthroughout theworld.

Optimizingandvalidatingthemolecularmethods

ThequalitativePCRanalyticalsensitivityinthefirst amplifi-cationwas101_{DNAcopies/␮Land,inthesecond,was10}2_DNA

copies/␮L.

ThedetectionthresholdofbothquantitativePCRsystems was100_{DNAcopies/␮L[quantificationcycle(Cq)∼35cycles].}

Therewerenodifferencesbetweentheresultsobtainedfrom standardvirusdilutionsinextractsofDNAofCNSnegativeto OvHV-2andinnucleasefreewater(Figs.1and2).

Sampleanalysis

Ofthebovine CNSsamplescollected from January 2012to March2014,4.2%(12/285)werepositiveforOvHV-2byqPCR. An additional four cases were confirmed in2009 and one in2007,totaling5.9%(17/290)positivity.ThequalitativePCR detected4.8%(14/290)positivesamples,and2.8%(8/290) pre-sentedcharacteristicminororgreatergradehistopathological changes(Fig.3).

TheagreementbetweenqPCR(themostsensitivemethod) andqualitativePCRwas82.4%(14/17)andbetweenqPCRand histologywas47.1%(8/17)(Table2).

Theviralloadofsamplesassociatedwithpathological evi-dencevariedfrom0.8×104 _to6.6×105_{DNAcopies/gtissue,}

andtheviralloadofsampleswithoutpathologyvariedfrom 0.9×103_to3.9×104_{DNAcopies/gtissue(Table2).}

SamplesofCNS,kidney,peripheralbloodleukocytes,nasal swab, eyes, and lymph nodes of the animal identified as 20428/13werecollected.Thetissueswerestoredseparately untilanalysis,topreventcrosscontamination,andanalyzed separately to evaluatevariations in positivity. All samples werepositivewithqualitativePCRand bothqPCRsystems.

Pathological changeswerefoundinthecarotidretemirabile

andinkidney.

Sequencing

Thirteenpositivesamplesfromcattleofdifferentregionsof Brazilwere sequenced(Fig.4).Theidentityamongsamples andbetweensamplesandsequencesobtainedfromthe Gen-Bankdatabaserangedfrom90.9to100%(Fig.4).

Discussion

MCFisgenerallydiagnosedbyhistory,epidemiologicaldata, clinical signs, and lesions observed during necropsy and microscopy.Occasionally,itisdiagnosedby immunodiagnos-ticsanddetectionofviralgenomicDNA.MalignantCatarrhal Fever diagnosis in Brazil is usuallymade byclinical signs, necropsy, andhistology.14–21 _{Suchdiagnosesarenotalways}

reliable,becauseMCFmaybeconfusedwithotherdiseases, resulting in under-diagnosis, since there are no character-istic macro- or microscopic lesions,and some lesions can be observedonlyupon post-mortemexamination. Molecu-larmethodsare reliableforMCFdiagnosis inante-mortem examinations(uncoagulatedblood,ocularandnasalswabs), providing early detection of the virus to avoid economic loss,disseminationofdisease withintheherd,and mortal-ity.Molecularmethodsallowpost-mortemdiagnosisinorgans andfluids.Molecularmethodsarehighlysensitiveandspecific andallowthediagnosisofMFCintheabsenceofclinicalsigns (latency), asmay exist inchronicdisease and inrecovered animals.22–24

BothqualitativeandquantitativePCRinthisstudyshowed highersensitivitythanreportedinpreviousstudies.9,24,25_The

useofSYBRGreenforOvHV-2diagnosisshowedthefeasibility ofthesemolecularmethodsinroutinelaboratorydiagnoses.

Amplification curve 105 ₁₀4 ₁₀3 ₁₀2 ₁₀1 ₁₀0 Select Zoom 35 5 16 399 14 899 13 399 11 899 10 399 8 899 7 399 5 899 4 399 2 899 1 399 –0 101 10 15 20 25 30 35 40 Error: 0 723 Efficiency: 1 994 Slope: –3 336 Ylntercept: 31.71 30 25 20 –1 –0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 Standard curve Cycles Log concentration Crossing point Fluorescence (465–510)

B

A

Fig.2–StandardcurveTaqMansystem,dilutionsinextractsofCNSofcattlenegativetoOvHV-2.(A)Serialdilutions100_–105 numbersofDNAcopies/␮L.(B)Specificvaluesofthereaction:efficacy=1.994;slope=−3.336.

Fig.3–HistopathologicalalterationsobservedinMCFintissuesofanimal798/07.(A)Carotidretemirabilewithmoderate mononuclearinflammatoryinfiltrationandhyalinizationofthetunicaadventitia(H&E200×magnification).(B)Cerebral cortexwithmoderatemononuclearperivascularcuffingingraymatterandmononuclearinfiltrateinmeninges

(non-purulentmeningo-encephalitis)(H&E200×magnification).(C)Thalamuswithintensemononuclearperivascular cuffingintheneuropil(H&E100×magnification).(D)Kidney.Renalcortexwithintensemononuclearinflammatory infiltrateandhyalinizationinthetunicamediaandadventitia(H&E100×magnification).

Table2–Sampleidentificationforyearcollecting,ageanimals(inmonths),numberofdeadorsickanimalsintheherd, quantificationcycle–Cq,DNAcopiespertissuegrambyqPCR,resultsofqualitativePCR,resultsofhistologyand country/statewherethesamplecamefrom.

Sample identification Year Age (months) Numberofdeador sickanimals/herd Quantification cycle(Cq)

DNAcopiesper tissuegram

Qualitative PCR

Histology County/state

798/07 2007 96 1/21 26.4 1.5×105 _{POS POS Assis/SP}

G355/09 2009 36 2/350 26.8 1.2×105 _{POS POS NI}a_/PE

G86/09 2009 48 3/530 24.6 4.6×105 _{POS POS NI}a_/PE

G251/09 2009 3 NIa _{26.5 1.5×10}5 _{POS POS LagoaD’Ouro/PE}

1398/09 2009 56 1/150 30.9 3.9×104 _{POS NEG Martinópolis/SP}

364/12 2012 38 NIa _{29.7 1.7×10}4 _{POS POS Socorro/SP}

11570/12 2012 84 NIa _{35.2 0.9×10}3 _{NEG NEG Cac¸apava/SP}

24221/12 2012 18 1/100 30.4 0.9×104 _{POS NEG RibeirãoPreto/SP}

20428/13 2013 36 NIa _{24.1 6.6×10}5 _{POS POS Bujari/AC}

5587/13 2013 48 NIa _{35.2 0.9×10}3 _{NEG NEG Franca/SP}

6414/13 2013 24 1/29 32.5 2.9×104 _{POS NEG Restinga/SP}

7983/13 2013 48 5/350 33.3 1.4×104 _{POS NEG Itirapuã/SP}

8011/13 2013 NIa _{12/3106 33.6 1.1×10}4 _{POS NEG Queiroz/SP}

8012/13 2013 48 1/397 28.9 3.9×104 _{POS POS Morungaba/SP}

8013/13 2013 5 1/254 33.5 1.0×104 _{POS NEG Itaguac¸u/ES}

25605/13 2013 48 1/350 33.7 0.8×104 _{POS POS SilvaJardim/RJ}

2062/14 2014 36 3/144 35.0 1.0×103 _{NEG NEG Itirapina/SP}

Fig.4–Locationofthesequencedsamples:A–798/07;B–G251/09;C–1398/09;D–364/12;E–24221/12;F–20428/13;G– 6414/13;H–7983/13;I–8012/13;J–8013/13;andK–25605/13.Phylogramofthenucleotidesequencesobtainedinthe currentstudyalignedwithOvHV-2sequencesavailableinGenBank.Thetreewasbuiltbytheneighbor-joiningalgorithm usingtheKimura2evolutionarymodelwith1000bootstrapreplicates.

Asexpected,thenumberofpositivesampleswashigherin bothqPCRanalyses(5.9%,17/290)thaninqualitativePCR(4.8% –14/290),reflectingdifferencesinanalyticalsensitivity.The sampleswith numbers of viral particles exceeding101_DNA

copies/␮L presented typical MCF histopathologies (2.8% – 8/290)inCNSandkidney (Fig.3).TheqPCR detectedtwice theSA-MCFcasesasfoundbyhistology, consideredagold standardmethod.Theseobservationsdemonstratethe impor-tanceofimplementingamoresensitive,specific,rapid,and safemoleculardiagnosismethod,allowingearlydiagnosisand easyOvHV-2detection.Thismethodallowsdetectionoflow viralloadsinarangeoforgansandfluids.

Thisresearchalsodrawsattentiontothe importanceof correctcollectionanddeliveryofmaterialforhistological diag-nosis.Inmostcasesapathologistdoesnotreceivethecarotid

retemirabiletissuenecessaryforMCFdiagnosis,making histo-logicconfirmationdifficultandleadingtounder-diagnosis.In thiscase,moleculardiagnosisisfundamental,duetoits abil-itytodetecttheagentinvariousorgansandfluids.Inaddition, theuseofbothmoleculartoolsandhistologyisusefulto differ-entiateactivefromlatentOvHV-2infection,consideringthat inflammatorylesionswithmononuclearinfiltrate(Fig.3)are relatedtothepresenceofaviralagent.

Mostofthefarmsonwhichpositiveanimalswerefound hadcattleandsheeppasturedtogether,whichwasariskfactor forSA-MCF.

The phylogenetic tree and the genetic identity among Braziliansamplesandwiththosefromotherpartsoftheworld showed nosignificantgenomic differences,suggestingthat theOvHV-2diagnosedinBraziliancattlewasgenetically sim-ilartothatfoundinothercountries.

TheseepidemiologicaldataareimportantforBrazilian Ani-malHealthauthoritiesplanningstrategiesforpreventionof MCFincattle.

Conflicts

of

interest

Theauthorsdeclarethatthereisnoconflictsofinterest.

Acknowledgements

Thanks toCoordenac¸ão de Aperfeic¸oamento de Pessoal de NívelSuperior(CAPES)oftheBrazilianMinistryofEducation andCultureforgrantingtheMaster’sscholarship,andtothe techniciansoftheCoordenadoriadeDefesaAgropecuáriado

EstadodeSãoPaulo(SecretaryofAgricultureandSupplyof SãoPauloState)forprovidingtheCNSsamples.

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