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Ortín et al. - 2025 - The Treatment of Contagious Ecthyma in Lambs with

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AcademicEditor:CliveJ.C.PhillipsReceived:10November2025Revised:4December2025Accepted:17December2025Published:20December2025Copyright:©2025bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license. CommunicationTheTreatmentofContagiousEcthymainLambswithaLocalAnaesthetic/AntisepticWoundFormulationLowersSerumAmyloidAResponsesAuroraOrtín1,2,* ,SergioVillanueva-Saz1,2 ,DeliaLacasta1,2 ,PeterAndrewWindsor3 ,AntonioFernández1 ,PabloQuilez1,2 ,HectorRuiz1,2 ,AlexGómez1,2,DavidGuallar1,2 andMartaRuizdeArcaute1,2 1AnimalPathologyDepartment,VeterinaryFacultyandInstitutoAgroalimentariodeAragón-IA2(UniversidaddeZaragoza-CITA),MiguelServet177,50013Zaragoza,Spain;svs@unizar.es(S.V.-S.);dlacasta@unizar.es(D.L.);afmedica@unizar.es(A.F.);pquilez@unizar.es(P.Q.);hectorruiz353@gmail.com(H.R.);alex.gomezcalvo@gmail.com(A.G.);davidguallarabellan2002@gmail.com(D.G.);martarda@unizar.es(M.R.d.A.)2RuminantClinicalService,VeterinaryFaculty,UniversityofZaragoza,MiguelServet177,50013Zaragoza,Spain3SydneySchoolofVeterinaryScience,TheUniversityofSydney,Camden,NSW2570,Australia;peter.windsor@sydney.edu.au*Correspondence:aortin@unizar.es;Tel.:+34-605319592SimpleSummaryContagiousecthyma(CE)isaskindiseaseofsmallruminantswithsignificanteconomicimpactandwelfareconcerns.VaccinationisthemainpreventivestrategyforCE,althoughinmanycountries,licencedvaccinesareunavailable.Treatmenttypicallyinvolvesan-tibioticstocontrolsecondaryinfections,increasingtheriskofantimicrobialresistance.Theobjectiveofthisstudywastoevaluatethetherapeuticeffectofanon-antibiotictopicalanaesthetic/antiseptictherapeuticformulation(Tri-Solfen®;T-S;MedicalEthics,Australia/MultiSolfen®;M-S;Dechra,UK)onlambswithcontagiousecthyma.Thecon-centrationofamarkerofsystemicinflammation,serumamyloidA(SAA),wasmeasuredduringtheclinicalphaseofCEinnaturallyandexperimentallyinfectedlambs,incohortseithertreatedornottreatedwiththeproduct.Intheexperimentalinfection,thetreatmentmodifiedtheSAAresponse,peakingearlierandatlowerlevelsthanincontrolsandshow-ingsignificantlylowervaluesatthecompletionoftheexperimentalperiodthanincontrols.Inthenaturaloutbreak,SAAlevelssignificantlydecreasedovertimeinthetreatedcohort,whereasincontrols,levelsremainedstableathighvalues.TheseresultsindicatethatthistopicalformulationreducessystemicinflammationinlambswithCE,providingsupportiveevidencethatthisisapromisingnon-antibiotictherapeuticalternativetocurrentpractice.AbstractContagiousecthyma(CE)isawidespread,highlycontagiouszoonoticskindiseaseofsmallruminantscausedbytheOrfvirus(ORFV),leadingtosubstantialeconomiclossesandwelfareconcerns.Thereisnospecifictreatment,withtopicalantisepticsandoralorparenteralantibioticsoftenadministeredforpreventingsecondaryinfections,riskingantimicrobialresistance.ThisstudyassessedtheeffectoftreatingCEinlambswithanantibiotic-freetopicalanaesthetic/antisepticformulation(Tri-Solfen®;T-S;MedicalEthics,Australia/MultiSolfen®;M-S;Dechra,UK).SerumamyloidA(SAA),amarkerofsystemicinflammation,wasmeasuredinbothexperimentallyandnaturallyinfectedlambsallocatedtotreatedanduntreatedgroups.Sampleswerecollectedpriorto(T0)andat2(T2),7(T7)and14(T14)dayspost-treatmentinexperimentallyinfectedlambsandatT0,10(T10)and Animals2026,16,17https://doi.org/10.3390/ani16010017 Animals2026,16,17 2of12 20(T20)dayspost-treatmentinnaturallyaffectedlambs.Intheexperimentalinfection,SAAconcentrationswerelowerinthetreatedgroupthanincontrolsatT7andsignificantlyloweratT14.Inthenaturaloutbreak,SAAconcentrationssignificantlydecreasedovertimeinthetreatedgroup,withaconsistenttrendtowardlowervaluesthanincontrols.ThesefindingsindicatethatthistherapeuticformulationreducessystemicinflammatoryresponsesinlambsaffectedbyCE,supportingitsuseasanalternativetoantibiotics.Keywords:orfvirus;contagiousecthyma;lambs;localantiseptic/anaestheticformulation;serumamyloidA 1.IntroductionContagiousecthyma(CE,Orf)isahighlycontagious,globallydistributedskindiseaseprimarilyaffectingsmallruminants,especiallyyounganimalsintheirfirstyearoflife[1].Thediseaseisanon-systemic,eruptiveskininfectioncausedbytheOrfvirus(ORFV),amemberofthefamilyPoxviridaeandgenusParapoxvirus[2].Clinically,CEischaracterisedbypapules,vesicles,pustulesandproliferativescabbylesions,mainlyofthemuzzleandlipmucosae,oftenextendingtotheoralmucosa,nostrils,earsandeyelidsand,lessfrequently,thefeet,genitalsandudder[1,3–6].CEisaself-limitingdisease,withlesionsusuallyresolvingwithin6–8weeks[4].However,ORFVencodesimmunomodulatoryproteinsthatinterferewiththehostimmuneresponse,favouringreinfectionsandpredisposinganimalstosecondaryinfections[7,8].Thisvirus-inducedimmunosuppressioncontributestotheriskofpersistenceoftheinfection,recurrenceafterlivevirusvaccinationandsecondarycomplicationsincludingbacterialcontaminationandinfection[4,6].OutbreaksofCEinfectionmaycausesubstantialanimalwelfareconcerns,withfrequentantibioticuseraisingtherisksofantimicrobialresistance.ORFVismainlytransmittedthroughskincutsorabrasions[9].Morbiditycanreach100%,withmortalityusually<5%,althoughinveryyounganimalswithsecondaryinfec-tions,upto90%mortalityhasbeenreported[10,11].CEcausessignificanteconomiclosses,particularlyinintensivehusbandrysystems,duetomortalityinyounganimals,reducedproductivitylinkedtodecreasedfoodintakecausedbypainfullesionsandinterferenceinsheepmarketingandtradingduetoclinicalsimilaritiestotransboundaryvesicularviraldiseases[6,12,13].AsORFVinfectionisalsoazoonosis,humansinclosecontactwithinfectedanimals,particularlyfarmers,veterinarians,shearersandslaughterhouseworkers,areoccasionallyinfected,typicallydevelopinglocalisedskinlesionsonthehands[6,14,15].AppropriatehygieneanddisinfectionmeasuresareessentialtopreventORFVinfec-tion.Thequarantineofnewlyintroducedanimalsandseparationofsickonesarerequired,aswellasthethoroughdisinfectionoffacilitiesandmaterials[4].OnceORFVentersandestablishesinaflock,eradicationisdifficult[4].Theviruscanpersistindryenvironments,remainingviableonvarioussurfacesandanimalby-productsforlongperiods.Immuno-compromisedorpersistentlyinfectedanimalsmaintaintheenvironmentalcirculationofORFV[16].VaccinationisthemainpreventivestrategyforCE,althoughinmanycountries,li-cencedvaccinesareunavailableforsheepandgoats[6,17,18].Existingvaccinesincludescab-basedandattenuatedtissueculturevaccines[8,19,20],althoughthesemayreverttovirulence.TheiruseisnotrecommendedonfarmswithoutahistoryofCE,astheycancontaminatetheenvironment[4].Despitetheseissues,liveCEvaccinesareusedwidelyinAustralia,wheretheysuccessfullysuppressthediseaseinmanyflocks[6,19].Asthedura-tionofprotectiveimmunityfromcurrentvaccinesisvariable,thedevelopmentofasafe,https://doi.org/10.3390/ani16010017 Animals2026,16,17 3of12 moreeffectiveandreadilyaccessibleORFVvaccineremainsapriority[1,17].PromisingprototypesofDNAandsubunitvaccinestargetingORFVB2LandORFVF1Lproteinshavebeendescribed[21–24],andcurrentresearchisfocusingongeneticmanipulationtodeleteORFVvirulencegenesandgenerateattenuatedvaccineswithstrongerimmunoprotectiveproperties[25,26].AsthereisnospecifictherapyforCE,currentapproachesincludestandardhygienepractices,localtopicalantisepticsandtopicalorparenteralantibiotics,administeredmainlytomanagetheriskofsecondaryinfections.Localantisepticsreportedincludesodiumpermanganateandsalicylicacid[27],potassiumpermanganateandboricacidointments[9],3%iodinesolution,gentianviolet[28]andhypochlorousacid[29].Traditionalherbalremediesincludingsesameorcastoroil,giantmilkweedjuiceandEuphorbiahavealsobeenused[4].AlthoughineffectiveagainstORFV[30],antibioticsappliedtopicallyorsystemicallyarewidelyusedtotreatsecondarybacterialinfections,contributingtotherisksofantimicrobialresistance(AMR)[13,31].WiththeincreasingglobalconcernsofAMRfromlivestockincludinginsmallruminantmedicine,thereisaneedtoexplorealternativestoantibiotictherapyforCE.Tri-Solfen®(T-S,MedicalEthics,Melbourne,Australia),laterregisteredandmarketedinEuropeasMultiSolfen®(M-S;Dechra,Northwich,UK),isawoundtherapeuticfor-mulationcombiningtwolocalanaesthetics(lidocaineandbupivacaine),adrenalineandanantiseptic(cetrimide)inagelmatrix.DevelopedinAustraliaforsurgicalhusbandryproceduresinsheep,itcreatesabarriereffect,blockingnociception,reducingpainandacceleratinghealing[32].Itwaslatershowntobeeffectiveintreatingoralmucosalle-sionsincattlewithfootandmouthdisease[33,34].ItslowpH(~2.7)mayalsoconfervirucidalactivity,potentiallyreducingORFVexpressionandtransmission.PreliminarytrialsinCE-affectedlambssuggestedreducedviralloadsintreatedanimals,althoughnodifferencesinclinicallesionprogressionwasobserved[18].Inexperimentallyinfectedlambs,T-Streatmentdidnotaffectweightgainorlesionprogression,possiblyduetothedeepepitheliallocationoftheinduced-CElesionsandtoaninadequatetreatmentprotocoladministeredtooearlyandwithtoofewapplications[31].However,inalaterfieldstudyinacommercialflock,whentheproducthadalreadybeenregisteredinEurope,repeatedM-Streatmentappliedaftervesicleeruptionandonthreeoccasionsreducedthenumberandseverityoflesionsintreatedversuscontrolanimals,suggestedasevidenceofefficacyandsupportingitsuseinfieldconditions[29].Acutephaseproteins(APPs)arealargefamilyofunrelatedproteins[35]withplasmaconcentrationsthatmayincrease(positiveAPPs)ordecrease(negativeAPPs)inresponsetoinflammation,infection,traumaorstress[36,37].Synthesisedmainlyintheliver,APPscontributetolimitingmicrobialgrowth,minimisingtissuedamageandrestoringhome-ostasis[38].Theirmeasurementhasdiagnostic,prognosticandmonitoringapplicationsininfectiousandinflammatoryconditions,aswellasinsubclinicaldiseasedetection[37,39].TheAPPprofileisspecies-specific;inruminants,haptoglobin(Hp)andserumamyloidA(SAA)arethemostrelevant,showingupto1000-foldincreasesinresponsetostim-uli[36,40,41].However,theSAAresponseinlambsisgenerallystrongerthanthatofHp[36].Hplevelsremainunchangedinlambsfollowingaversivestimuli,includingtaildockingandcastration,whereasthisAPPisaneffectivemarkerformonitoringtheseprocessesincattle[40].OurgroupalsohasevidenceofastrongerSAAresponsethanthatofHpinfeedlotlambs[42].Inlambs,SAAshowsaparticularlyrapidandintenseresponse,withpeakswithin24–48hafterstimulusandnormalisationwithin4–7daysifnofurtherchallengeoccurs[36,43].ThisstudyevaluatedtheevolutionoftheSAAresponseduringtheclinicalphaseofCEinnaturallyandexperimentallyinfectedlambs,incohortseithertreatedornottreatedhttps://doi.org/10.3390/ani16010017 Animals2026,16,17 4of12 withM-S/T-S.TheobjectivewastoassesswhetherthepotentialtherapeuticeffectofthisformulationonCE-affectedlambscouldalsobereflectedintheserumlevelsofthemostrelevantAPPinlambs.2.MaterialsandMethodsAllproceduresweresupervisedandapprovedbytheEthicsCommitteeforAnimalExperimentsoftheUniversityofZaragoza(referencePI33/21).AnimalcareandusecompliedwiththeSpanishPolicyforAnimalProtection(RD53/2013),whichalignswiththeEuropeanUnionDirective2010/63ontheprotectionofanimalsusedforexperimentalandotherscientificpurposes.Twoexperimentalstudieswereconducted:onewithlambsexperimentallyinfectedwithORFVandanotheronacommercialsheepfarmnaturallyaffectedbyaCEoutbreak.Inbothstudies,animalswereallocatedintotwocohorts:(i)treatedwithM-SorT-Sor(ii)untreated(control).SAAwasmeasuredintreatedanduntreatedlambsthroughouttheexperimentalperiod.2.1.StudyinLambsExperimentallyInfectedwithORFVTheexperimentaldesignpreviouslydescribed[31]forevaluatingtheeffectofT-StreatmentinlambsexperimentallyinfectedwithORFVwasfollowedinthisstudy.Briefly,50healthy,four-day-oldmalelambswererecruitedfromaLacaunedairysheepfarmthathadnotexperiencedCEoutbreaksinthepreviousthreeyears.AlllambsweretestedbyPCRandELISAandconfirmednegativeforORFVbeforebeingtransferredtothefacilitiesoftheVeterinaryFacultyofZaragoza.Oncethere,theywererandomlyallocatedintotwoindependentandfullyisolatedpens,with25lambsineach.Alllambswereexperimentallyinfectedwithaliquotsof1mLofinoculumcontaining104TCID50(mediantissuecultureinfectiousdose)ofORFV.Theinoculumwasadminis-teredusinganintradermalinoculationgun,distributedacrossthelips,gumsandpalate[31].Briefly,anaverageof17inoculationswereadministeredforeachlamb(1.7mL/animal),splitbetweentheright,leftandcentralupperlips;right,leftandcentrallowerlips;andtheupperandloweraspectsoftheinteriorofthemouth(gumsand/orpalate).Eightdaysafterinfection,oncethefirstCElesionsappeared,lambsfrompen2(treatmentgroup)weretreatedwithT-S(Tri-Solfen®,MedicalEthics,Melbourne,Australia),spraying1.5mLoftheproductwithadosinggunoverthelipsandinsidethemouth,ensuringthecompletecoverageofalllesions.Treatmentwasrepeatedthreedayslater(11dayspost-inoculation)intheaffectedareasoftheface(Table1).Lambsfrompen1(controlgroup)didnotreceiveanytreatment.Table1.TreatmentandsamplingscheduleinlambsexperimentallyinfectedwithOrfvirus. DaysPost-Treatment −8EI†−1023714 SeverityofthelesionsInitiallesionsT-Streatment§1T-S2T-SBloodsampling‡T0T2T7T14 †EI=Experimentalinfection.§T-S=Tri-Solfen®;1T-S=firstdoseoftreatment;2T-S=seconddoseoftreatment.Controlgroupanimalsremaineduntreated.‡Bloodsampleswerecollectedfromtreatedanduntreatedlambspriortotreatment(T0)and2days(T2),7days(T7)and14days(T14)aftertheapplicationofthefirstdoseoftreatment.https://doi.org/10.3390/ani16010017 Animals2026,16,17 5of12 Forthisstudy,bloodsampleswithoutanticoagulantwerecollectedbyjugularvenipuncturefromlambsinbothgroupsatfourtimepoints:thedaybeforethefirsttreatment(T0)andat2(T2),7(T7)and14(T14)daysafterthefirsttreatment(Table1).Serawereobtained,aliquotedandstoredat–20◦CuntiltheanalysisofSAAconcentrations.2.2.StudyinLambsNaturallyAffectedbyContagiousEcthymaThisstudywaspartofalargerexperimentthatevaluatedtheeffectofM-StreatmentinacommercialsheepfarmaffectedbyaCEoutbreak[29].Briefly,inthatexperiment,150Lacauneneonatallambsaged25–30dayswereselectedonthebasisofclinicalpresenta-tionwithskinororallesionsconsistentwithCE,andinfectionwasconfirmedbyPCR.Thelambswererandomlydividedintothreecohorts(groupsC,DandE)of50animalseach.LambsingroupCweretreatedwithM-S(Dechra,Northwich,UK),spraying1.5mLoftheproductwithadosinggunoverthelipsandinsidethemouth,ensuringthecompletecoverageofalllesions.Thetreatmentwasappliedonthreeoccasionswith3-dayintervals(Table2).AnimalsingroupDweretreateddailywithhypochlorousacidusingthesametechniqueasingroupC,withlambsingroupEservingasuntreatedcontrols.Table2.Treatmentandsamplingscheduleinlambsnaturallyaffectedbycontagiousecthyma. DaysPost-Treatment −10481020 SeverityofthelesionsArangeofskinandorallesionsM-Streatment§1M-S2M-S3M-SBloodsampling‡T0T10T20 §M-S=Multi-Solfen®;1M-S=firstdoseoftreatment;2M-S=seconddoseoftreatment;3M-S=thirddoseoftreatment.Controlgroupanimalsremaineduntreated.‡Bloodsampleswerecollectedfromtreatedanduntreatedlambspriortotreatment(T0)and10days(T10)and20days(T20)aftertheapplicationofthefirstdoseoftreatment.Forthepresentstudy,12lambsfromgroupC(M-Streated)and12lambsfromgroupE(control)wererandomlyselected.Bloodsampleswithoutanticoagulantwerecollectedbyjugularvenipunctureatthreetimepoints:thedaybeforethefirsttreatment(T0)andat10(T10)and20(T20)daysafterthefirsttreatment(Table2).Sampleswereprocessedtoobtainsera,aliquotedandstoredat–20◦CuntilthemeasurementofSAAconcentrations.2.3.SerumAmyloidA(SAA)ConcentrationSAAconcentrationsweremeasuredusingasolid-phasesandwichELISAkit(PHASE™SerumAmyloidAAssay,TrideltaDevelopmentLtd.,Maynooth,Ireland)followingthemanufacturer’sinstructions.AbsorbancewasreadwithaMultiskanMSmicroplatereader(Labsystems,Helsinki,Finland).Sampleconcentrationswerecalculatedbyinterpolationfromthecalibrationcurvegeneratedwiththekitcalibrators,adjustingforthecorrespondingdilutionfactor.Allsampleswereanalysedinduplicate,andthemeanvaluewasused.Sampleswithabsorbancevaluesoutsidethecalibrationrangewerere-analysedafterfurtherdilutiontobringthemwithinrange.Theintra-andinter-assaycoefficientsofvariation(CVs)were5.0%and11.4%,respectively.Theassaysensitivitywas0.3µg/mL.2.4.StatisticalAnalysisofResultsDatawereanalysedusingIBMSPSSStatisticsversion26.0(Armonk,NY,USA).Non-parametrictestswereapplied,asthevariabledidnotmeetnormalityassumptions.TheFriedmantestwasusedtoevaluatetheeffectoftimeonSAAconcentrations.DifferencesbetweenT-S/M-S-treatedanduntreatedgroupsateachtimepointwereassessedwiththehttps://doi.org/10.3390/ani16010017 Animals2026,16,17 6of12 Mann–Whitneytest.Inallanalyses,differenceswereconsideredstatisticallysignificantatp<0.05.3.Results3.1.StudyinLambsExperimentallyInfectedwithORFVTheevolutionofSAAconcentrationsinlambsexperimentallyinfectedwithORFVisdisplayed(Figure1),comparingthegrouptreatedwithT-Sandtheuntreatedcontrolgroup.ThemedianvaluestogetherwiththefirstandthirdquartilesforeachsamplingtimepointandthepercentageofreductioninSAAintreatedlambscomparedtountreatedonesarealsodisplayed(Table3). Figure1.SerumamyloidA(SAA)concentrationovertimeinlambsexperimentallyinfectedwithORFVthatweretreatedornottreated(n=25foreachgroup)withTri-Solfen®(T-S).SampleswerecollectedatT0(priortotreatment),T2(2days),T7(7days)andT14(14days)afterthefirsttreatment.Differentletters(a,b)indicatesignificantdifferencesbetweendifferentsamplingtimes.Differentletters(x,y)indicatesignificantdifferencesbetweengroups.Table3.Medianvaluesand1stand3rdquartilesofserumamyloidA(SAA)concentrationsatdifferentsamplingtimesinlambsexperimentallyinfectedwithORFV,treatedornottreatedwithTri-Solfen®(T-S),andpercentageofreductioninSAAintreatedlambscomparedtountreatedones. SAA(ng/mL) SamplingTime§Group†n=25Median1stQuartile3rdQuartile%ofReduction T0T-S2297.561045.204836.25C4107.551320.029028.42T2T-S4747.102610.0010,649.00---C3837.47558.2315,721.45T7T-S10,475.452410.0024,240.750.29C21,075.053377.9577,487.62T14T-S4058.222046.4527,375.0089.11C37,283.776166.51154,459.75 §T0=priortotreatment;2days(T2),7days(T7)and14days(T14)aftertheapplicationofthefirstdoseoftreatment.†T-S=groupoflambstreatedwithTri-Solfen®;C=control.https://doi.org/10.3390/ani16010017 Animals2026,16,17 7of12 Inthecontrolgroup,SAAlevelsincreasedovertime,reachingthehighestvalue,37,283(6166.51–154,459.75)ng/mL,attheendoftheexperimentalperiod(T14).Atthistimepoint,SAAconcentrationsweresignificantlyhigherthanatT2andT0(p=0.030andp=0.013,respectively).NosignificantdifferenceswereobservedbetweenT0andT2,althoughthevalueatT7wassignificantlyhigherthanthoseatbothT0(p=0.024)andT2(p=0.020).InthegrouptreatedwithT-S,SAAlevelsalsoincreasedovertimebutonlyduringthefirsthalfoftheexperimentalperiod.ThepeakoccurredatT7,withavalueof10,475(2410–24,240.7)ng/mL,whichwaslowerthanthepeakrecordedinthecontrolgroupatT14.Inthetreatedgroup,SAAconcentrationatT7washigherthanthatatT0,T2andT14,althoughsignificantdifferenceswereonlydetectedbetweenT7andT0(p=0.001).Nosig-nificantdifferenceswerefoundbetweengroupsatT0orT2.However,SAAconcentrationsatT7andT14werelowerintheT-S-treatedgroupcomparedwiththecontrolgroup(theywerereducedby50.29%and89.11%,respectively),withthedifferencereachingsignificanceatT14(p=0.041).3.2.StudyinLambsNaturallyAffectedbyContagiousEcthymaTheevolutionofSAAconcentrationsinlambsnaturallyaffectedbyCEisshown(Figure2),comparingthegrouptreatedwithM-Sandtheuntreatedcontrolgroup.ThemedianvaluestogetherwiththefirstandthirdquartilesforeachsamplingtimepointandthepercentageofreductioninSAAintreatedlambscomparedtountreatedonesarealsodisplayed(Table4). Figure2.SerumamyloidA(SAA)concentrationsovertimeinlambsnaturallyaffectedbycontagiousecthyma,treatedornottreated(n=12foreachgroup)withMulti-Solfen®(M-S).Sampleswerecollectedpriortotreatment(T0)andat10(T10)and20(T20)daysafterthefirsttreatment.Differentletters(a,b)indicatesignificantdifferencesbetweendifferentsamplingtimes.https://doi.org/10.3390/ani16010017 Animals2026,16,17 8of12 Table4.Medianvaluesand1stand3rdquartilesofserumamyloidA(SAA)concentrationsatdif-ferentsamplingtimesinlambsnaturallyaffectedbycontagiousecthyma,treatedornottreatedwithMulti-Solfen®(M-S),andpercentageofreductioninSAAintreatedlambscomparedtountreatedones. SAA(ng/mL) SamplingTime§Group†n=12Median1stQuartile3rdQuartile%ofReduction T0M-S39,456.5030,996.17105,669.65C29,843.0016,205.50111,879.62T10M-S14,300.305726.0222,225.2045C26,269.516,551.7545,727.95T20M-S12,326.002799.0223,381.5056.75C28,499.008699.9971,160.02 §T0=priortotreatment;10days(T10)and20days(T20)aftertheapplicationofthefirstdoseoftreatment.†M-C=groupoflambstreatedwithMulti-Solfen®;C=controlgroup(untreatedlambs).Inthecontrolgroup,SAAconcentrationatthebeginningofthisstudywasveryhigh,29,843(16,205.50–111,879.62)ng/mL,andremainedsimilaratallsamplingtimes,withnosignificantdifferencesdetectedovertime.InthegroupoflambstreatedwithM-S,SAAconcentrationatT0wasalsoveryhigh,39,456(30,996.17–105,669.65)ng/mL,butdecreasedaftertreatment.ValuesatT10andT20weresignificantlylowerthanthatatT0(p=0.030forboth).Whencomparingthetwogroups,nosignificantdifferencesweredetectedatanysamplingtime.However,atrendtowardslowervalueswasobservedintheM-S-treatedgroupcomparedwithcontrolsatT10(p=0.061)andT20(p=0.081),andSAAlevelswerereducedby45%and56.75%atT10andT20,respectively.4.DiscussionCEisagloballydistributed,highlycontagiouszoonoticviralskindisease,causingsignificanteconomiclossesinaffectedsheepandgoatfarms.Onlyafewregisteredvaccinesexist,andthesearenotreadilyavailableinmanyEuropeanandAsiancountries.ThereiscurrentlynoeffectivetreatmentforCE,andlocalantisepticsandantibioticsarefrequentlyused,increasingtheriskofAMR.T-S/M-Shasbeenproposedasapotentialnon-antibiotictherapeuticalternativeforthisdisease.Inaninitialstudywith50experimentallyinfectedlambs,T-StreatmentdidnotaffecttheclinicalprogressionofCElesions,aresultattributedtoaninadequatetreatmentprotocoladministeredtooearlyandwithtoofewapplications[31].Bycontrast,inasubsequentfieldstudyinacommercialflockaffectedbyaCEoutbreak,M-Swasappliedaftervesicleeruptionandonthreeoccasions,resultinginfewerlambswithORFV-associatedlesionsandmilderlesionscomparedwithuntreatedanimals.ThisfindingsuggestedthattopicalM-Scanbeeffectiveunderfieldconditionsiftheappropriatetherapeuticprotocolisapplied[29].ThepresentstudyaimedtoprovidefurtherevidenceofthetherapeuticeffectofT-S/M-SinCE-affectedlambs.SAA,themostrelevantAPPinlambs,wasanalysedinbloodsamplescollectedfromlambsparticipatingintwopreviouslypublishedstudies[29,31].OurfindingintheexperimentallyinfectedlambswasthatT-StreatmentmodifiedtheSAAresponse,peakingearlierandatlowerlevelsthanincontrols.WhilstSAAlevelsinthecontrolgroupincreasedprogressively,reachingthemaximumattheendoftheexperimentalperiod(T14),inthegrouptreatedwithT-S,thepeakoccurredearlier,atT7,followedbyadecline.Consequently,atT14,treatedlambshadsignificantlylowerSAAconcentrationsthancontrols.Althoughitwaspreviouslyreportedthattherewerenoclinicaldifferencesbetweengroups[31],ourSAAdatafromthisstudysuggestapotentialhttps://doi.org/10.3390/ani16010017 Animals2026,16,17 9of12 therapeuticeffectofT-SontheinflammatoryresponseinCEexperimentallyinducedlambs,highlightingtheimportanceofoptimisingtreatmentprotocolstoenabletheproducttoachieveitsfullpotential.Insupportofthisaim,inthepublishedfieldstudyinwhichM-Swasappliedaftervesicleeruptioninnaturallyinfectedlambsandonthreeoccasions[29],clinicaldifferencesbetweengroupswerereported.ThepresentanalysisofSAAconcentrationsinasubsampleofanimalsfromthatstudyprovidesadditionalevidenceforthetherapeuticpotentialofM-SinCEunderfieldconditions.Innaturallyaffectedlambs,baselineSAAconcentrationsatT0werealreadyveryhigh,reflectingthefactthatCElesionsweremoreadvancedatthetimeofthefirstsamplingthanintheexperimentaltrial.Inthisgroup,M-StreatmentwasassociatedwithasignificantdecreaseinSAAvaluesatT10andT20comparedwithT0,whereasincontrols,levelsremainedstableathighvalues.Althoughintergroupcomparisonsdidnotreachstatisticalsignificance,aconsistenttrendtowardslowerSAAconcentrationswasobservedintreatedlambsatbothlatertimepoints,suggestingthatthesamplesizemaynothavebeensufficienttodetectdifferenceswithgreaterstatisticalpower.TheseresultsindicatethatM-StreatmentmayreducethesystemicSAAresponse,consistentwiththetherapeuticsuppressionoftheinflammatoryresponse,innaturallyaffectedtreatedlambsaswell,inagreementwiththeclinicalfindings.OurresultsofareducedSAAresponsefollowingT-S/M-StherapyareconsistentwiththoseobservedinlambstreatedwithT-Sfollowingtaildocking[43].Inbothofourstudies,SAAconcentrationsweremuchhigherthanthebaselinevaluesreportedforlambsintheliterature[44,45],althoughthiswasexpected,sinceSAAincreasesmarkedlyinresponsetoinflammation,infectionortrauma.ElevatedT0valueswerealsoobservedinbothstudies,likelyreflectingthefactthatCElesionsweredevelopingorhadalreadyappearedandtriggeredthesystemicresponsepriortothefirstsampling.TheSAAresponsetypicallyriseswithin24–48hafterthetriggeringevent[38].However,thisinitialincreasewasmuchmorepronouncedinnaturallyaffectedlambs,likelyduetomoreadvancedlesiondevelopmentatT0comparedwiththeexperimentalstudy.Inbothstudies,SAAconcentrationsdidnotreturntobaselineduringtheobservationperiod,despitereportsintheliteraturedescribingarapidnormalisationwithin4–7daysifnofurtherstimulioccur[36,43].InCE,lesionsmaypersistfor6–8weeks[4,5],pos-siblyduetorecurrentreinfections[1,4]or,morelikely,theseverepathologicalnatureoftheproliferativedermatitisinCElesions,characterisedbytheballooningofthecyto-plasmofhyperplasticbasalepithelialcellsdeeplyembeddedinthevicinityofthedermis,whichislikelytobeongoingandrefractorytorapidhealing[6].AlthoughT-S/M-ShasdemonstratedvirucidalactivityandacapacitytoreduceviralloadinCElesions[18],thepenetrationofactivesintothedeeperbasalepitheliummaybeinsufficienttobecurative,enablingongoinginfectiontosustaintheSAAresponse.Multiple-doseregimensandlongertreatmentperiodsmaythereforebenecessary,potentiallyextendingforatleast4weeks,thetimeusuallyrequiredforlesionresolution.5.ConclusionsAlthoughT-S/M-ScannotbeexpectedtofullyeliminateORFVfromproliferativebasalepidermallesions,ourfindingssuggestthatitisapromisingtherapyforCE,capableofreducinginflammation,improvinganimalwelfareandhelpingcontrolsecondaryinfec-tionswithoutpromotingAMR.Further,ourresultsalsosupportthepotentialofSAAasabiomarkerformonitoringacutephaseresponsesduringCEandforassessingdiseasepro-gression.ThesefindingsshouldencouragefurtherresearchintooptimisingCEtreatmentprotocolsandtovalidateSAAasapracticaltoolformonitoringfieldoutbreaks.https://doi.org/10.3390/ani16010017 Animals2026,16,17 10of12 AuthorContributions:Conceptualisation,P.A.W.,D.L.andA.O.;methodology,S.V.-S.,A.O.andD.L.;software,A.F.;validation,S.V.-S.andA.O.;formalanalysis,A.F.;investigation,P.Q.,H.R.,A.G.,D.G.andM.R.d.A.;resources,P.Q.,H.R.andM.R.d.A.;datacuration,A.F.;writing—originaldraftpreparation,A.O.;writing—reviewandediting,D.L.andP.A.W.;visualisation,A.F.andA.O.;supervision,D.L.;projectadministration,D.L.;fundingacquisition,P.A.W.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwassupportedbyfundingandproductsfromtheAustraliancompanyMedicalEthics.ThisworkwasalsosupportedbytheAragónGovernment(A15_20RandA15_23R).InstitutionalReviewBoardStatement:AlltheproceduresweresupervisedandapprovedbytheEthicsAdvisoryCommissionforAnimalExperimentation(no.PI33/21),theBiosafetyCommitteeandtheOccupationalRiskPreventionUnitoftheUniversityofZaragoza,inaccordancewithcurrentregulationsregardingtheseprocedures,aspects:R.D.53/2013(whichalignswiththeEuropeanUnionDirective2010/63ontheprotectionofanimalsusedforexperimentalandotherscientificpurposes),Law31/1995,R.D.664/1997,R.D.1299/2006.InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Therawdatasupportingtheconclusionsofthisarticlewillbemadeavailablebytheauthorsonrequest.Acknowledgments:TheauthorswouldliketoacknowledgetheinternsoftheRuminantClinicalServiceoftheVeterinaryFacultyofZaragozainvolvedinthisstudy.TheywouldalsoliketothankMariaÁngelesLostaoforhercollaborationinthelaboratorywork.ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest,althoughP.A.W.hasprovidedoccasionaladvisoryservicetoMedicalEthicsAustralia.Thefunderwasnotinvolvedinthestudydesign,collection,analysis,interpretationofdata,thewritingofthisarticleorthedecisiontosubmititforpublication.References1.Bala,J.A.;Balakrishnan,K.N.;Abdullah,A.A.;Mohamed,R.;Haron,A.W.;Jesse,F.F.A.;Nordin,M.M.;Mohd-Azmi,M.L.There-emergingoforfvirusinfection:Acallforsurveillance,vaccinationandeffectivecontrolmeasures.Microb.Pathog.2018,120,55–63.[CrossRef]2.Bergqvist,C.;Kurban,M.;Abbas,O.Orfvirusinfection.Rev.Med.Virol.2017,9,1932.[CrossRef]3.McElroy,M.C.;Bassett,H.F.Thedevelopmentoforallesionsinlambsnaturallyinfectedwithorfvirus.Vet.J.2007,174,663–664.[CrossRef]4.Nandi,S.;De,U.K.;Chowdhury,S.Currentstatusofcontagiousecthymaororfdiseaseingoatandsheep—Aglobalperspective.SmallRum.Res.2011,96,1366–1370.[CrossRef]5.Fleming,S.B.;Wise,L.M.;Mercer,A.A.MoleculargeneticanalysisofOrfVirus:Apoxvirusthathasadaptedtoskin.Viruses2015,7,1505–1539.[CrossRef][PubMed]6.Windsor,P.A.;Nampanya,S.;Tagger,A.;Keonam,K.;Gerasimova,M.;Putthana,V.;Bush,R.D.;Khounsy,S.Isorfinfectionarisktoexpandinggoatproductionindevelopingcountries?AstudyfromLaoPDR.SmallRum.Res.2017,154,123–128.[CrossRef]7.Rohde,J.;Emschermann,F.;Knittle,M.R.;Rziha,H.J.OrfvirusinterfereswithMHCclassIsurfaceexpressionbytargetingvesiculartransportandGolgi.BMCVet.Res.2012,8,114.[CrossRef][PubMed]8.Bukar,A.M.;Jesse,F.F.A.;Abdullah,C.A.C.;Noordin,M.M.;Lawan,Z.;Mangga,H.K.;Balakrishnnan,K.N.;Azmi,M.L.M.Immunomodulatorystrategiesforparapoxivirus:CurrentstatusandfutureapproachesforthedevelopmentofvaccinesagainstOrfvirusinfection.Vaccines2021,9,1341.[CrossRef][PubMed]9.Spyrou,V.;Valiakos,G.Orfvirusinfectioninsheeporgoats.Vet.Microbiol.2015,181,178–182.[CrossRef]10.Gumbrell,R.C.;McGregor,D.A.Outbreakofseverfatalorfinlambs.Vet.Rec.1997,141,150–151.[CrossRef]11.Hosamani,M.;Scagliarini,A.;Bhanuprakash,V.;McInnes,C.J.;Singh,R.K.Orf:Anupdateoncurrentresearchandfutureperspectives.Expert.Rev.Anti.Infect.Ther.2009,7,879–893.[CrossRef][PubMed]12.Lovatt,F.M.;Barker,W.J.W.;Brown,D.;Spooner,R.K.Case-controlstudyoforfinpreweanedlambsandanassessmentofthefinancialimpactofthedisease.Vet.Rec.2012,170,673.[CrossRef]13.Lawan,Z.;Bala,J.A.;Bukar,A.M.;Balakrishnan,K.N.;Mangga,H.K.;Abdullah,F.F.J.;Noor-din,M.M.;Mohd-Azmi,M.L.Contagiousecthyma:Howseriousisthediseaseworldwide?Anim.HealthRes.Rev.2021,22,40–55.[CrossRef]https://doi.org/10.3390/ani16010017 Animals2026,16,17 11of12 14.Maor,D.;Yu,L.L.;Brand,R.Acaseoforfdiseaseinapatientwithscleroderma.JAADCaseRep.2017,3,155–157.[CrossRef]15.Kassa,T.AReviewonHumanOrf:ANeglectedViralZoonosis.Res.Rep.Trop.Med.2021,12,153–172.[CrossRef]16.Yeruham,I.;Perl,S.;Abraham,A.OrfInfectioninFourSheepFlocks.Vet.J.2000,160,74–76.[CrossRef]17.Lacasta,D.;Ferrer,L.M.;Ramos,J.J.;González,J.M.;Ortín,A.;Fthenakis,G.C.Vaccinationschedulesinsmallruminantsfarms.Vet.Microbiol.2015,181,34–36.[CrossRef]18.Lacasta,D.;Reina,R.;RuizdeArcaute,M.;Ferrer,L.M.;Benito,A.A.;Tejedor,M.T.;Echeverria,I.;Ruiz,H.;Cardenas,S.M.;Windsor,P.A.EffectofatopicalformulationoninfectiveviralloadinlambsnaturallyinfectedwithOrfvirus.Vet.Med.Res.Rep.2021,12,149–158.[CrossRef]19.Pye,D.Vaccinationofsheepwithcellculturegrownorfvirus.Aust.Vet.J.1990,67,182–186.[CrossRef][PubMed]20.Musser,J.M.B.;Taylor,C.A.;Guo,J.;Tizard,I.R.;Walker,J.W.Developmentofacontagiousecthymavaccineforgoats.Am.J.Vet.Res.2008,69,1366–1370.[CrossRef][PubMed]21.Zhao,K.;He,W.;Gao,W.;Lu,H.;Han,T.;Li,J.;Zhang,X.;Zhang,B.;Wang,G.;Su,G.;etal.OrfvirusDNAvaccinesexpressingORFV011andORFV059chimericproteinenhancesimmunogenicity.Virol.J.2011,8,562.[CrossRef]22.Yogisharadhya,R.;Bhanuprakash,V.;Kumar,A.;Mondal,M.;Schivachandra,S.B.ComparativesequenceandstructuralanalysisofIndianorfvirusesbasedonmajorenvelopeimmuno-dominantprotein(F1L),anhomologueofpoxviralp35/H3protein.Gene2018,663,72–82.[CrossRef][PubMed]23.Wassie,T.;Fammei,Z.;Jiang,X.;Liu,G.;Girmay,S.;Min,Z.;Chenhui,L.;Bo,D.D.;Ahmed,S.RecombinantB21andkisspeptin-54DNAvaccineinducesimmunityagainstorfvirusandinhibitsspermatogenesisinrats.Sci.Rep.2019,9,11.[CrossRef][PubMed]24.Wang,Y.;Sun,S.;Zhao,K.;Du,L.;Wang,X.;He,W.;Gao,F.;Song,D.;Guan,J.OrfvirusDNAprime-proteinbooststrategyissuperiortoadenovirus-basedvaccinationinmiceandsheep.Front.Immunol.2023,14,1077938.[CrossRef]25.Zhu,Z.;Qu,G.;Du,J.;Wang,C.;Chen,Y.;Shen,Z.;Zhou,Z.;Yin,C.;Chen,X.Constructionandcharacterizationofacontagiousecthymavirusdoublé-genedelectionstrainandevaluationofitspotentialasalive-attenuatedvaccineingoat.Front.Immunol.2022,13,961287.[CrossRef]26.Shen,Z.;Liu,B.;Zhu,Z.;Du,J.;Zhou,Z.;Pan,C.;Chen,Y.;Yin,C.;Luo,Y.;Li,H.;etal.Constructionofatriple-genedeletionmutantoforfvirusandevaluationofitssafety,immunogenicityandprotectiveefficacy.Vaccines2023,11,909.[CrossRef]27.Tontis,A.;Koning,H.;Kaderli,R.;Walter,L.Outbreaksofcontagiousecthymaintwoflocksofsheepandaherdofgoats.Schweiz.Arch.Tierheilkd.1981,123,19–28.28.Adedeji,A.;Adole,J.;Chima,N.;Maguda,A.;Dyek,D.;Jambol,A.;Anefu,E.;Shallmizhili,J.;Luka,P.ContagiousecthymainthreeflocksofgoatsinJos-southLGA,PlateauState,Nigeria.SokotoJ.Vet.Sci.2018,16,107.[CrossRef]29.Gómez,A.;Lacasta,D.;Tejedor,M.T.;RuizdeArcaute,M.;Ramos,J.J.;Ruiz,H.;Ortín,A.;Villanueva-Saz,S.;Reina,R.;Quilez,P.;etal.UseofalocalanaestheticandantisepticwoundformulationforthetreatmentoflambsnaturallyinfectedwithOrfvirus.Vet.Microbiol.2024,292,110037.[CrossRef]30.Greig,A.;Linklator,K.;Clark,W.Persistentorfinaram.Vet.Rec.1984,115,49.[CrossRef]31.Lacasta,D.;Ríos,M.;RuizdeArcaute,M.;Ortín,A.;Ramos,J.J.;Villanueva-Saz,S.;Tejedor,M.T.;Ruiz,H.;Borobia,M.;Reina,R.;etal.Useofalocalanaesthetic/antisepticformulationforthetreatmentoflambsexperimentallyinfectedwithOrfvirus.Animals2023,13,2962.[CrossRef][PubMed]32.Roberts,C.D.;Windsor,P.A.Innovativepainmanagementsolutionsinanimalsmayprovideimprovedwoundpainreductionduringdebridementinhumans:Anopinióninformedbyveterinaryliterature.Int.WoundJ.2019,16,968–973.[CrossRef][PubMed]33.Windsor,P.A.;Khounsy,S.;Earp,F.;MacPhillamy,I.;Young,J.;Bush,R.managingwelfareandantimicrobia-resistanceissuesintreatingfoot-and-mouthdiseaselesions:Anewtherapeuticapproach.Vet.Med.Res.Rep.2020,11,99–107.[CrossRef]34.Lendzele,S.S.;Mavoungou,J.F.;Burinyuy,K.A.;Armel,K.A.;Dickmu,S.J.;Young,J.R.;Thomson,P.C.;Windsor,P.A.EfficacyandapplicationofanoveltopicalanaestheticwoundformulationfortreatingcattlewithFoot-and-Mouthdisease:AfieldtrialinCameroon.Transbound.Emerg.Dis.2021,68,2531–2542.[CrossRef]35.Ceciliani,F.;Ceron,J.J.;Eckersall,P.D.;Sauerwein,H.Acutephaseproteinsinruminants.J.Proteomics2012,75,4207–4231.[CrossRef][PubMed]36.Petersen,H.H.;Nielsen,J.P.;Heegaard,P.M.H.Applicationofacutephaseproteinmeasurementsinveterinaryclinicalchemistry.Vet.Res.2004,35,163–187.[CrossRef]37.Eckersall,P.D.Proteins,proteomics,andthedysproteinemias.InClinicaBiochemistryofDomesticAnimals,6thed.;Kaneko,J.J.,Harvey,J.W.,Bruss,M.L.,Eds.;ElsevierAcademicPress:Davis,CA,USA,2008;pp.117–155,ISBN9780123704917.38.Tothova,C.;Nagy,O.;Kovac,G.Acutephaseproteinsandtheiruseinthediagnosisofdiseasesinruminants:Areview.Vet.Med.2014,59,163–180.[CrossRef]39.Saco,Y.;Bassols,A.Acutephaseproteinsincattleandswine:Areview.Vet.Clin.Pathol.2023,52,50–63.[CrossRef]40.Murata,H.;Shimada,N.;Yoshioka,M.Currentresearchonacutephaseproteinsinveterinarydiagnosis:Anoverview.Vet.J.2004,168,28–40.[CrossRef]https://doi.org/10.3390/ani16010017 Animals2026,16,17 12of12 41.Reczy´nska,D.;Zalewska,M.;Czopowicz,M.;Kaba,J.;Zwierzchowski,L.;Bagnicka,E.Acutephaseproteinlevelsasanauxiliarytoolindiagnosingviraldiseasesinruminants:Areview.Viruses2018,10,502.[CrossRef]42.Navarro,T.;González,J.M.;Ramos,J.J.;Marca,M.C.;Figliola,L.;RuizdeArcaute,M.;Borobia,M.;Ortín,A.Impactofstressonhealthandfinalweightinfatteninglambs.Animals2020,10,1274.[CrossRef]43.Ferrer,L.M.;Lacasta,D.;Ortín,A.;Ramos,J.J.;Tejedor,M.T.;Borobia,M.;Pérez,M.;Castells,E.;RuizdeArcaute,M.;Ruiz,H.;etal.Impactodatopicalanaesthesiawoundmanagementformulationonpain,inflammationandreductionofsecondaryinfectionsaftertaildockinginlambs.Animals2020,10,1255.[CrossRef]44.Lepherd,M.L.;Canfield,P.F.;Hunt,G.B.;Bosward,K.L.Haematological,biochemicalandselectedacutephaseproteinreferenceintervalsforweanedfemaleMerinolambs.Aust.Vet.J.2009,87,5–11.[CrossRef]45.Dinler,C.;Tuna,G.E.;Ay,E.;Ulutas,B.;Voyvoda,H.y.;Ulutas,P.A.ReferenceintervalsforserumamyloidA,haptoglobin,ceruloplasmin,andfibrinogeninapparentlyhealthyneonatallambs.Vet.Clin.Path.2020,49,484–490.[CrossRef]Disclaimer/Publisher’sNote:Thestatements,opinionsanddatacontainedinallpublicationsaresolelythoseoftheindividualauthor(s)andcontributor(s)andnotofMDPIand/ortheeditor(s).MDPIand/ortheeditor(s)disclaimresponsibilityforanyinjurytopeopleorpropertyresultingfromanyideas,methods,instructionsorproductsreferredtointhecontent.https://doi.org/10.3390/ani16010017
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AcademicEditor:CliveJ.C.PhillipsReceived:10November2025Revised:4December2025Accepted:17December2025Published:20December2025Copyright:©2025bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommonsAttribution(CCBY)license. CommunicationTheTreatmentofContagiousEcthymainLambswithaLocalAnaesthetic/AntisepticWoundFormulationLowersSerumAmyloidAResponsesAuroraOrtín1,2,* ,SergioVillanueva-Saz1,2 ,DeliaLacasta1,2 ,PeterAndrewWindsor3 ,AntonioFernández1 ,PabloQuilez1,2 ,HectorRuiz1,2 ,AlexGómez1,2,DavidGuallar1,2 andMartaRuizdeArcaute1,2

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-Saz1,2 ,DeliaLacasta1,2 ,PeterAndrewWindsor3 ,AntonioFernández1 ,PabloQuilez1,2 ,HectorRuiz1,2 ,AlexGómez1,2,DavidGuallar1,2 andMartaRuizdeArcaute1,2 1AnimalPathologyDepartment,VeterinaryFacultyandInstitutoAgroalimentariodeAragón-IA2(UniversidaddeZaragoza-CITA),MiguelServet177,50013Zaragoza,Spain;svs@unizar.es(S.V.-S.);dlacasta@unizar.es(D.L.);afmedica@unizar.es(A.F.);pquilez@unizar.es(P.Q.);hectorruiz353@gmail.com(H.R.);alex.gomezcalvo@gmail.com(A.G.);davidguallarabellan2002@gmail.com(D.G.);martarda@unizar.es(M.R.d.A.)2RuminantClinicalService,VeterinaryFaculty,UniversityofZaragoza,MiguelServet177,50013Zaragoza,Spain3SydneySchoolofVeterinaryScience,TheUniversityofSydney,Camden,NSW2570,Australia;peter.windsor@sydney.edu.au*Correspondence:aortin@unizar.es;Tel.:+34-605319592SimpleSummaryContagiousecthyma(CE)isaskindiseaseofsmallruminantswithsignificanteconomicimpactandwelfareconcerns.VaccinationisthemainpreventivestrategyforCE,althoughinmanycountries,licencedvaccinesareunavailable.Treatmenttypicallyinvolvesan-tibioticstocontrolsecondaryinfections,increasingtheriskofantimicrobialresistance.Theobjectiveofthisstudywastoevaluatethetherapeuticeffectofanon-antibiotictopicalanaesthetic/antiseptictherapeuticformulation(Tri-Solfen®;T-S;MedicalEthics,Australia/MultiSolfen®;M-S;Dechra,UK)onlambswithcontagiousecthyma.Thecon-centrationofamarkerofsystemicinflammation,serumamyloidA(SAA),wasmeasuredduringtheclinicalphaseofCEinnaturallyandexperimentallyinfectedlambs,incohortseithertreatedornottreatedwiththeproduct.Intheexperimentalinfection,thetreatmentmodifiedtheSAAresponse,peakingearlierandatlowerlevelsthanincontrolsandshow-ingsignificantlylowervaluesatthecompletionoftheexperimentalperiodthanincontrols.Inthenaturaloutbreak,SAAlevelssignificantlydecreasedovertimeinthetreatedcohort,whereasincontrols,levelsremainedstableathighvalues.TheseresultsindicatethatthistopicalformulationreducessystemicinflammationinlambswithCE,providingsupportiveevidencethatthisisapromisingnon-antibiotictherapeuticalternativetocurrentpractice.AbstractContagiousecthyma(CE)isawidespread,highlycontagiouszoonoticskindiseaseofsmallruminantscausedbytheOrfvirus(ORFV),leadingtosubstantialeconomiclossesandwelfareconcerns.Thereisnospecifictreatment,withtopicalantisepticsandoralorparenteralantibioticsoftenadministeredforpreventingsecondaryinfections,riskingantimicrobialresistance.ThisstudyassessedtheeffectoftreatingCEinlambswithanantibiotic-freetopicalanaesthetic/antisepticformulation(Tri-Solfen®;T-S;MedicalEthics,Australia/MultiSolfen®;M-S;Dechra,UK).SerumamyloidA(SAA),amarkerofsystemicinflammation,wasmeasuredinbothexperimentallyandnaturallyinfectedlambsallocatedtotreatedanduntreatedgroups.Sampleswerecollectedpriorto(T0)andat2(T2),7(T7)and14(T14)dayspost-treatmentinexperimentallyinfectedlambsandatT0,10(T10)and

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totreatedanduntreatedgroups.Sampleswerecollectedpriorto(T0)andat2(T2),7(T7)and14(T14)dayspost-treatmentinexperimentallyinfectedlambsandatT0,10(T10)and Animals2026,16,17https://doi.org/10.3390/ani16010017

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Animals2026,16,17 2of12 20(T20)dayspost-treatmentinnaturallyaffectedlambs.Intheexperimentalinfection,SAAconcentrationswerelowerinthetreatedgroupthanincontrolsatT7andsignificantlyloweratT14.Inthenaturaloutbreak,SAAconcentrationssignificantlydecreasedovertimeinthetreatedgroup,withaconsistenttrendtowardlowervaluesthanincontrols.ThesefindingsindicatethatthistherapeuticformulationreducessystemicinflammatoryresponsesinlambsaffectedbyCE,supportingitsuseasanalternativetoantibiotics.Keywords:orfvirus;contagiousecthyma;lambs;localantiseptic/anaestheticformulation;serumamyloidA

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fectedbyCE,supportingitsuseasanalternativetoantibiotics.Keywords:orfvirus;contagiousecthyma;lambs;localantiseptic/anaestheticformulation;serumamyloidA 1.IntroductionContagiousecthyma(CE,Orf)isahighlycontagious,globallydistributedskindiseaseprimarilyaffectingsmallruminants,especiallyyounganimalsintheirfirstyearoflife[1].Thediseaseisanon-systemic,eruptiveskininfectioncausedbytheOrfvirus(ORFV),amemberofthefamilyPoxviridaeandgenusParapoxvirus[2].Clinically,CEischaracterisedbypapules,vesicles,pustulesandproliferativescabbylesions,mainlyofthemuzzleandlipmucosae,oftenextendingtotheoralmucosa,nostrils,earsandeyelidsand,lessfrequently,thefeet,genitalsandudder[1,3–6].CEisaself-limitingdisease,withlesionsusuallyresolvingwithin6–8weeks[4].However,ORFVencodesimmunomodulatoryproteinsthatinterferewiththehostimmuneresponse,favouringreinfectionsandpredisposinganimalstosecondaryinfections[7,8].Thisvirus-inducedimmunosuppressioncontributestotheriskofpersistenceoftheinfection,recurrenceafterlivevirusvaccinationandsecondarycomplicationsincludingbacterialcontaminationandinfection[4,6].OutbreaksofCEinfectionmaycausesubstantialanimalwelfareconcerns,withfrequentantibioticuseraisingtherisksofantimicrobialresistance.ORFVismainlytransmittedthroughskincutsorabrasions[9].Morbiditycanreach100%,withmortalityusually<5%,althoughinveryyounganimalswithsecondaryinfec-tions,upto90%mortalityhasbeenreported[10,11].CEcausessignificanteconomiclosses,particularlyinintensivehusbandrysystems,duetomortalityinyounganimals,reducedproductivitylinkedtodecreasedfoodintakecausedbypainfullesionsandinterferenceinsheepmarketingandtradingduetoclinicalsimilaritiestotransboundaryvesicularviraldiseases[6,12,13].AsORFVinfectionisalsoazoonosis,humansinclosecontactwithinfectedanimals,particularlyfarmers,veterinarians,shearersandslaughterhouseworkers,areoccasionallyinfected,typicallydevelopinglocalisedskinlesionsonthehands[6,14,15].AppropriatehygieneanddisinfectionmeasuresareessentialtopreventORFVinfec-tion.Thequarantineofnewlyintroducedanimalsandseparationofsickonesarerequired,aswellasthethoroughdisinfectionoffacilitiesandmaterials[4].OnceORFVentersandestablishesinaflock,eradicationisdifficult[4].Theviruscanpersistindryenvironments,remainingviableonvarioussurfacesandanimalby-productsforlongperiods.Immuno-compromisedorpersistentlyinfectedanimalsmaintaintheenvironmentalcirculationofORFV[16].VaccinationisthemainpreventivestrategyforCE,althoughinmanycountries,li-cencedvaccinesareunavailableforsheepandgoats[6,17,18].Existingvaccinesincludescab-basedandattenuatedtissueculturevaccines[8,19,20],althoughthesemayreverttovirulence.TheiruseisnotrecommendedonfarmswithoutahistoryofCE,astheycancontaminatetheenvironment[4].Despitetheseissues,liveCEvaccinesareusedwidelyinAustralia,wheretheysuccessfullysuppressthediseaseinmanyflocks[6,19].Asthedura-tionofprotectiveimmunityfromcurrentvaccinesisvariable,thedevelopmentofasafe,https://doi.org/10.3390/ani16010017

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Animals2026,16,17 3of12

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Animals2026,16,17 3of12 moreeffectiveandreadilyaccessibleORFVvaccineremainsapriority[1,17].PromisingprototypesofDNAandsubunitvaccinestargetingORFVB2LandORFVF1Lproteinshavebeendescribed[21–24],andcurrentresearchisfocusingongeneticmanipulationtodeleteORFVvirulencegenesandgenerateattenuatedvaccineswithstrongerimmunoprotectiveproperties[25,26].AsthereisnospecifictherapyforCE,currentapproachesincludestandardhygienepractices,localtopicalantisepticsandtopicalorparenteralantibiotics,administeredmainlytomanagetheriskofsecondaryinfections.Localantisepticsreportedincludesodiumpermanganateandsalicylicacid[27],potassiumpermanganateandboricacidointments[9],3%iodinesolution,gentianviolet[28]andhypochlorousacid[29].Traditionalherbalremediesincludingsesameorcastoroil,giantmilkweedjuiceandEuphorbiahavealsobeenused[4].AlthoughineffectiveagainstORFV[30],antibioticsappliedtopicallyorsystemicallyarewidelyusedtotreatsecondarybacterialinfections,contributingtotherisksofantimicrobialresistance(AMR)[13,31].WiththeincreasingglobalconcernsofAMRfromlivestockincludinginsmallruminantmedicine,thereisaneedtoexplorealternativestoantibiotictherapyforCE.Tri-Solfen®(T-S,MedicalEthics,Melbourne,Australia),laterregisteredandmarketedinEuropeasMultiSolfen®(M-S;Dechra,Northwich,UK),isawoundtherapeuticfor-mulationcombiningtwolocalanaesthetics(lidocaineandbupivacaine),adrenalineandanantiseptic(cetrimide)inagelmatrix.DevelopedinAustraliaforsurgicalhusbandryproceduresinsheep,itcreatesabarriereffect,blockingnociception,reducingpainandacceleratinghealing[32].Itwaslatershowntobeeffectiveintreatingoralmucosalle-sionsincattlewithfootandmouthdisease[33,34].ItslowpH(~2.7)mayalsoconfervirucidalactivity,potentiallyreducingORFVexpressionandtransmission.PreliminarytrialsinCE-affectedlambssuggestedreducedviralloadsintreatedanimals,althoughnodifferencesinclinicallesionprogressionwasobserved[18].Inexperimentallyinfectedlambs,T-Streatmentdidnotaffectweightgainorlesionprogression,possiblyduetothedeepepitheliallocationoftheinduced-CElesionsandtoaninadequatetreatmentprotocoladministeredtooearlyandwithtoofewapplications[31].However,inalaterfieldstudyinacommercialflock,whentheproducthadalreadybeenregisteredinEurope,repeatedM-Streatmentappliedaftervesicleeruptionandonthreeoccasionsreducedthenumberandseverityoflesionsintreatedversuscontrolanimals,suggestedasevidenceofefficacyandsupportingitsuseinfieldconditions[29].Acutephaseproteins(APPs)arealargefamilyofunrelatedproteins[35]withplasmaconcentrationsthatmayincrease(positiveAPPs)ordecrease(negativeAPPs)inresponsetoinflammation,infection,traumaorstress[36,37].Synthesisedmainlyintheliver,APPscontributetolimitingmicrobialgrowth,minimisingtissuedamageandrestoringhome-ostasis[38].Theirmeasurementhasdiagnostic,prognosticandmonitoringapplicationsininfectiousandinflammatoryconditions,aswellasinsubclinicaldiseasedetection[37,39].TheAPPprofileisspecies-specific;inruminants,haptoglobin(Hp)andserumamyloidA(SAA)arethemostrelevant,showingupto1000-foldincreasesinresponsetostim-uli[36,40,41].However,theSAAresponseinlambsisgenerallystrongerthanthatofHp[36].Hplevelsremainunchangedinlambsfollowingaversivestimuli,includingtaildockingandcastration,whereasthisAPPisaneffectivemarkerformonitoringtheseprocessesincattle[40].OurgroupalsohasevidenceofastrongerSAAresponsethanthatofHpinfeedlotlambs[42].Inlambs,SAAshowsaparticularlyrapidandintenseresponse,withpeakswithin24–48hafterstimulusandnormalisationwithin4–7daysifnofurtherchallengeoccurs[36,43].ThisstudyevaluatedtheevolutionoftheSAAresponseduringtheclinicalphaseofCEinnaturallyandexperimentallyinfectedlambs,incohortseithertreatedornottreatedhttps://doi.org/10.3390/ani16010017

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Animals2026,16,17 4of12

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Animals2026,16,17 4of12 withM-S/T-S.TheobjectivewastoassesswhetherthepotentialtherapeuticeffectofthisformulationonCE-affectedlambscouldalsobereflectedintheserumlevelsofthemostrelevantAPPinlambs.2.MaterialsandMethodsAllproceduresweresupervisedandapprovedbytheEthicsCommitteeforAnimalExperimentsoftheUniversityofZaragoza(referencePI33/21).AnimalcareandusecompliedwiththeSpanishPolicyforAnimalProtection(RD53/2013),whichalignswiththeEuropeanUnionDirective2010/63ontheprotectionofanimalsusedforexperimentalandotherscientificpurposes.Twoexperimentalstudieswereconducted:onewithlambsexperimentallyinfectedwithORFVandanotheronacommercialsheepfarmnaturallyaffectedbyaCEoutbreak.Inbothstudies,animalswereallocatedintotwocohorts:(i)treatedwithM-SorT-Sor(ii)untreated(control).SAAwasmeasuredintreatedanduntreatedlambsthroughouttheexperimentalperiod.2.1.StudyinLambsExperimentallyInfectedwithORFVTheexperimentaldesignpreviouslydescribed[31]forevaluatingtheeffectofT-StreatmentinlambsexperimentallyinfectedwithORFVwasfollowedinthisstudy.Briefly,50healthy,four-day-oldmalelambswererecruitedfromaLacaunedairysheepfarmthathadnotexperiencedCEoutbreaksinthepreviousthreeyears.AlllambsweretestedbyPCRandELISAandconfirmednegativeforORFVbeforebeingtransferredtothefacilitiesoftheVeterinaryFacultyofZaragoza.Oncethere,theywererandomlyallocatedintotwoindependentandfullyisolatedpens,with25lambsineach.Alllambswereexperimentallyinfectedwithaliquotsof1mLofinoculumcontaining104TCID50(mediantissuecultureinfectiousdose)ofORFV.Theinoculumwasadminis-teredusinganintradermalinoculationgun,distributedacrossthelips,gumsandpalate[31].Briefly,anaverageof17inoculationswereadministeredforeachlamb(1.7mL/animal),splitbetweentheright,leftandcentralupperlips;right,leftandcentrallowerlips;andtheupperandloweraspectsoftheinteriorofthemouth(gumsand/orpalate).Eightdaysafterinfection,oncethefirstCElesionsappeared,lambsfrompen2(treatmentgroup)weretreatedwithT-S(Tri-Solfen®,MedicalEthics,Melbourne,Australia),spraying1.5mLoftheproductwithadosinggunoverthelipsandinsidethemouth,ensuringthecompletecoverageofalllesions.Treatmentwasrepeatedthreedayslater(11dayspost-inoculation)intheaffectedareasoftheface(Table1).Lambsfrompen1(controlgroup)didnotreceiveanytreatment.Table1.TreatmentandsamplingscheduleinlambsexperimentallyinfectedwithOrfvirus.

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asoftheface(Table1).Lambsfrompen1(controlgroup)didnotreceiveanytreatment.Table1.TreatmentandsamplingscheduleinlambsexperimentallyinfectedwithOrfvirus. DaysPost-Treatment −8EI†−1023714 SeverityofthelesionsInitiallesionsT-Streatment§1T-S2T-SBloodsampling‡T0T2T7T14 †EI=Experimentalinfection.§T-S=Tri-Solfen®;1T-S=firstdoseoftreatment;2T-S=seconddoseoftreatment.Controlgroupanimalsremaineduntreated.‡Bloodsampleswerecollectedfromtreatedanduntreatedlambspriortotreatment(T0)and2days(T2),7days(T7)and14days(T14)aftertheapplicationofthefirstdoseoftreatment.https://doi.org/10.3390/ani16010017

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Animals2026,16,17 5of12 Forthisstudy,bloodsampleswithoutanticoagulantwerecollectedbyjugularvenipuncturefromlambsinbothgroupsatfourtimepoints:thedaybeforethefirsttreatment(T0)andat2(T2),7(T7)and14(T14)daysafterthefirsttreatment(Table1).Serawereobtained,aliquotedandstoredat–20◦CuntiltheanalysisofSAAconcentrations.2.2.StudyinLambsNaturallyAffectedbyContagiousEcthymaThisstudywaspartofalargerexperimentthatevaluatedtheeffectofM-StreatmentinacommercialsheepfarmaffectedbyaCEoutbreak[29].Briefly,inthatexperiment,150Lacauneneonatallambsaged25–30dayswereselectedonthebasisofclinicalpresenta-tionwithskinororallesionsconsistentwithCE,andinfectionwasconfirmedbyPCR.Thelambswererandomlydividedintothreecohorts(groupsC,DandE)of50animalseach.LambsingroupCweretreatedwithM-S(Dechra,Northwich,UK),spraying1.5mLoftheproductwithadosinggunoverthelipsandinsidethemouth,ensuringthecompletecoverageofalllesions.Thetreatmentwasappliedonthreeoccasionswith3-dayintervals(Table2).AnimalsingroupDweretreateddailywithhypochlorousacidusingthesametechniqueasingroupC,withlambsingroupEservingasuntreatedcontrols.Table2.Treatmentandsamplingscheduleinlambsnaturallyaffectedbycontagiousecthyma. DaysPost-Treatment −10481020 SeverityofthelesionsArangeofskinandorallesionsM-Streatment§1M-S2M-S3M-SBloodsampling‡T0T10T20

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ffectedbycontagiousecthyma. DaysPost-Treatment −10481020 SeverityofthelesionsArangeofskinandorallesionsM-Streatment§1M-S2M-S3M-SBloodsampling‡T0T10T20 §M-S=Multi-Solfen®;1M-S=firstdoseoftreatment;2M-S=seconddoseoftreatment;3M-S=thirddoseoftreatment.Controlgroupanimalsremaineduntreated.‡Bloodsampleswerecollectedfromtreatedanduntreatedlambspriortotreatment(T0)and10days(T10)and20days(T20)aftertheapplicationofthefirstdoseoftreatment.Forthepresentstudy,12lambsfromgroupC(M-Streated)and12lambsfromgroupE(control)wererandomlyselected.Bloodsampleswithoutanticoagulantwerecollectedbyjugularvenipunctureatthreetimepoints:thedaybeforethefirsttreatment(T0)andat10(T10)and20(T20)daysafterthefirsttreatment(Table2).Sampleswereprocessedtoobtainsera,aliquotedandstoredat–20◦CuntilthemeasurementofSAAconcentrations.2.3.SerumAmyloidA(SAA)ConcentrationSAAconcentrationsweremeasuredusingasolid-phasesandwichELISAkit(PHASE™SerumAmyloidAAssay,TrideltaDevelopmentLtd.,Maynooth,Ireland)followingthemanufacturer’sinstructions.AbsorbancewasreadwithaMultiskanMSmicroplatereader(Labsystems,Helsinki,Finland).Sampleconcentrationswerecalculatedbyinterpolationfromthecalibrationcurvegeneratedwiththekitcalibrators,adjustingforthecorrespondingdilutionfactor.Allsampleswereanalysedinduplicate,andthemeanvaluewasused.Sampleswithabsorbancevaluesoutsidethecalibrationrangewerere-analysedafterfurtherdilutiontobringthemwithinrange.Theintra-andinter-assaycoefficientsofvariation(CVs)were5.0%and11.4%,respectively.Theassaysensitivitywas0.3µg/mL.2.4.StatisticalAnalysisofResultsDatawereanalysedusingIBMSPSSStatisticsversion26.0(Armonk,NY,USA).Non-parametrictestswereapplied,asthevariabledidnotmeetnormalityassumptions.TheFriedmantestwasusedtoevaluatetheeffectoftimeonSAAconcentrations.DifferencesbetweenT-S/M-S-treatedanduntreatedgroupsateachtimepointwereassessedwiththehttps://doi.org/10.3390/ani16010017

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Animals2026,16,17 6of12 Mann–Whitneytest.Inallanalyses,differenceswereconsideredstatisticallysignificantatp<0.05.3.Results3.1.StudyinLambsExperimentallyInfectedwithORFVTheevolutionofSAAconcentrationsinlambsexperimentallyinfectedwithORFVisdisplayed(Figure1),comparingthegrouptreatedwithT-Sandtheuntreatedcontrolgroup.ThemedianvaluestogetherwiththefirstandthirdquartilesforeachsamplingtimepointandthepercentageofreductioninSAAintreatedlambscomparedtountreatedonesarealsodisplayed(Table3). Figure1.SerumamyloidA(SAA)concentrationovertimeinlambsexperimentallyinfectedwithORFVthatweretreatedornottreated(n=25foreachgroup)withTri-Solfen®(T-S).SampleswerecollectedatT0(priortotreatment),T2(2days),T7(7days)andT14(14days)afterthefirsttreatment.Differentletters(a,b)indicatesignificantdifferencesbetweendifferentsamplingtimes.Differentletters(x,y)indicatesignificantdifferencesbetweengroups.Table3.Medianvaluesand1stand3rdquartilesofserumamyloidA(SAA)concentrationsatdifferentsamplingtimesinlambsexperimentallyinfectedwithORFV,treatedornottreatedwithTri-Solfen®(T-S),andpercentageofreductioninSAAintreatedlambscomparedtountreatedones. SAA(ng/mL) SamplingTime§Group†n=25Median1stQuartile3rdQuartile%ofReduction T0T-S2297.561045.204836.25C4107.551320.029028.42T2T-S4747.102610.0010,649.00---C3837.47558.2315,721.45T7T-S10,475.452410.0024,240.750.29C21,075.053377.9577,487.62T14T-S4058.222046.4527,375.0089.11C37,283.776166.51154,459.75 §T0=priortotreatment;2days(T2),7days(T7)and14days(T14)aftertheapplicationofthefirstdoseoftreatment.†T-S=groupoflambstreatedwithTri-Solfen®;C=control.https://doi.org/10.3390/ani16010017

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ays(T7)and14days(T14)aftertheapplicationofthefirstdoseoftreatment.†T-S=groupoflambstreatedwithTri-Solfen®;C=control.https://doi.org/10.3390/ani16010017 Animals2026,16,17 7of12 Inthecontrolgroup,SAAlevelsincreasedovertime,reachingthehighestvalue,37,283(6166.51–154,459.75)ng/mL,attheendoftheexperimentalperiod(T14).Atthistimepoint,SAAconcentrationsweresignificantlyhigherthanatT2andT0(p=0.030andp=0.013,respectively).NosignificantdifferenceswereobservedbetweenT0andT2,althoughthevalueatT7wassignificantlyhigherthanthoseatbothT0(p=0.024)andT2(p=0.020).InthegrouptreatedwithT-S,SAAlevelsalsoincreasedovertimebutonlyduringthefirsthalfoftheexperimentalperiod.ThepeakoccurredatT7,withavalueof10,475(2410–24,240.7)ng/mL,whichwaslowerthanthepeakrecordedinthecontrolgroupatT14.Inthetreatedgroup,SAAconcentrationatT7washigherthanthatatT0,T2andT14,althoughsignificantdifferenceswereonlydetectedbetweenT7andT0(p=0.001).Nosig-nificantdifferenceswerefoundbetweengroupsatT0orT2.However,SAAconcentrationsatT7andT14werelowerintheT-S-treatedgroupcomparedwiththecontrolgroup(theywerereducedby50.29%and89.11%,respectively),withthedifferencereachingsignificanceatT14(p=0.041).3.2.StudyinLambsNaturallyAffectedbyContagiousEcthymaTheevolutionofSAAconcentrationsinlambsnaturallyaffectedbyCEisshown(Figure2),comparingthegrouptreatedwithM-Sandtheuntreatedcontrolgroup.ThemedianvaluestogetherwiththefirstandthirdquartilesforeachsamplingtimepointandthepercentageofreductioninSAAintreatedlambscomparedtountreatedonesarealsodisplayed(Table4). Figure2.SerumamyloidA(SAA)concentrationsovertimeinlambsnaturallyaffectedbycontagiousecthyma,treatedornottreated(n=12foreachgroup)withMulti-Solfen®(M-S).Sampleswerecollectedpriortotreatment(T0)andat10(T10)and20(T20)daysafterthefirsttreatment.Differentletters(a,b)indicatesignificantdifferencesbetweendifferentsamplingtimes.https://doi.org/10.3390/ani16010017

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Animals2026,16,17 8of12 Table4.Medianvaluesand1stand3rdquartilesofserumamyloidA(SAA)concentrationsatdif-ferentsamplingtimesinlambsnaturallyaffectedbycontagiousecthyma,treatedornottreatedwithMulti-Solfen®(M-S),andpercentageofreductioninSAAintreatedlambscomparedtountreatedones. SAA(ng/mL) SamplingTime§Group†n=12Median1stQuartile3rdQuartile%ofReduction T0M-S39,456.5030,996.17105,669.65C29,843.0016,205.50111,879.62T10M-S14,300.305726.0222,225.2045C26,269.516,551.7545,727.95T20M-S12,326.002799.0223,381.5056.75C28,499.008699.9971,160.02

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29,843.0016,205.50111,879.62T10M-S14,300.305726.0222,225.2045C26,269.516,551.7545,727.95T20M-S12,326.002799.0223,381.5056.75C28,499.008699.9971,160.02 §T0=priortotreatment;10days(T10)and20days(T20)aftertheapplicationofthefirstdoseoftreatment.†M-C=groupoflambstreatedwithMulti-Solfen®;C=controlgroup(untreatedlambs).Inthecontrolgroup,SAAconcentrationatthebeginningofthisstudywasveryhigh,29,843(16,205.50–111,879.62)ng/mL,andremainedsimilaratallsamplingtimes,withnosignificantdifferencesdetectedovertime.InthegroupoflambstreatedwithM-S,SAAconcentrationatT0wasalsoveryhigh,39,456(30,996.17–105,669.65)ng/mL,butdecreasedaftertreatment.ValuesatT10andT20weresignificantlylowerthanthatatT0(p=0.030forboth).Whencomparingthetwogroups,nosignificantdifferencesweredetectedatanysamplingtime.However,atrendtowardslowervalueswasobservedintheM-S-treatedgroupcomparedwithcontrolsatT10(p=0.061)andT20(p=0.081),andSAAlevelswerereducedby45%and56.75%atT10andT20,respectively.4.DiscussionCEisagloballydistributed,highlycontagiouszoonoticviralskindisease,causingsignificanteconomiclossesinaffectedsheepandgoatfarms.Onlyafewregisteredvaccinesexist,andthesearenotreadilyavailableinmanyEuropeanandAsiancountries.ThereiscurrentlynoeffectivetreatmentforCE,andlocalantisepticsandantibioticsarefrequentlyused,increasingtheriskofAMR.T-S/M-Shasbeenproposedasapotentialnon-antibiotictherapeuticalternativeforthisdisease.Inaninitialstudywith50experimentallyinfectedlambs,T-StreatmentdidnotaffecttheclinicalprogressionofCElesions,aresultattributedtoaninadequatetreatmentprotocoladministeredtooearlyandwithtoofewapplications[31].Bycontrast,inasubsequentfieldstudyinacommercialflockaffectedbyaCEoutbreak,M-Swasappliedaftervesicleeruptionandonthreeoccasions,resultinginfewerlambswithORFV-associatedlesionsandmilderlesionscomparedwithuntreatedanimals.ThisfindingsuggestedthattopicalM-Scanbeeffectiveunderfieldconditionsiftheappropriatetherapeuticprotocolisapplied[29].ThepresentstudyaimedtoprovidefurtherevidenceofthetherapeuticeffectofT-S/M-SinCE-affectedlambs.SAA,themostrelevantAPPinlambs,wasanalysedinbloodsamplescollectedfromlambsparticipatingintwopreviouslypublishedstudies[29,31].OurfindingintheexperimentallyinfectedlambswasthatT-StreatmentmodifiedtheSAAresponse,peakingearlierandatlowerlevelsthanincontrols.WhilstSAAlevelsinthecontrolgroupincreasedprogressively,reachingthemaximumattheendoftheexperimentalperiod(T14),inthegrouptreatedwithT-S,thepeakoccurredearlier,atT7,followedbyadecline.Consequently,atT14,treatedlambshadsignificantlylowerSAAconcentrationsthancontrols.Althoughitwaspreviouslyreportedthattherewerenoclinicaldifferencesbetweengroups[31],ourSAAdatafromthisstudysuggestapotentialhttps://doi.org/10.3390/ani16010017

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Animals2026,16,17 9of12 therapeuticeffectofT-SontheinflammatoryresponseinCEexperimentallyinducedlambs,highlightingtheimportanceofoptimisingtreatmentprotocolstoenabletheproducttoachieveitsfullpotential.Insupportofthisaim,inthepublishedfieldstudyinwhichM-Swasappliedaftervesicleeruptioninnaturallyinfectedlambsandonthreeoccasions[29],clinicaldifferencesbetweengroupswerereported.ThepresentanalysisofSAAconcentrationsinasubsampleofanimalsfromthatstudyprovidesadditionalevidenceforthetherapeuticpotentialofM-SinCEunderfieldconditions.Innaturallyaffectedlambs,baselineSAAconcentrationsatT0werealreadyveryhigh,reflectingthefactthatCElesionsweremoreadvancedatthetimeofthefirstsamplingthanintheexperimentaltrial.Inthisgroup,M-StreatmentwasassociatedwithasignificantdecreaseinSAAvaluesatT10andT20comparedwithT0,whereasincontrols,levelsremainedstableathighvalues.Althoughintergroupcomparisonsdidnotreachstatisticalsignificance,aconsistenttrendtowardslowerSAAconcentrationswasobservedintreatedlambsatbothlatertimepoints,suggestingthatthesamplesizemaynothavebeensufficienttodetectdifferenceswithgreaterstatisticalpower.TheseresultsindicatethatM-StreatmentmayreducethesystemicSAAresponse,consistentwiththetherapeuticsuppressionoftheinflammatoryresponse,innaturallyaffectedtreatedlambsaswell,inagreementwiththeclinicalfindings.OurresultsofareducedSAAresponsefollowingT-S/M-StherapyareconsistentwiththoseobservedinlambstreatedwithT-Sfollowingtaildocking[43].Inbothofourstudies,SAAconcentrationsweremuchhigherthanthebaselinevaluesreportedforlambsintheliterature[44,45],althoughthiswasexpected,sinceSAAincreasesmarkedlyinresponsetoinflammation,infectionortrauma.ElevatedT0valueswerealsoobservedinbothstudies,likelyreflectingthefactthatCElesionsweredevelopingorhadalreadyappearedandtriggeredthesystemicresponsepriortothefirstsampling.TheSAAresponsetypicallyriseswithin24–48hafterthetriggeringevent[38].However,thisinitialincreasewasmuchmorepronouncedinnaturallyaffectedlambs,likelyduetomoreadvancedlesiondevelopmentatT0comparedwiththeexperimentalstudy.Inbothstudies,SAAconcentrationsdidnotreturntobaselineduringtheobservationperiod,despitereportsintheliteraturedescribingarapidnormalisationwithin4–7daysifnofurtherstimulioccur[36,43].InCE,lesionsmaypersistfor6–8weeks[4,5],pos-siblyduetorecurrentreinfections[1,4]or,morelikely,theseverepathologicalnatureoftheproliferativedermatitisinCElesions,characterisedbytheballooningofthecyto-plasmofhyperplasticbasalepithelialcellsdeeplyembeddedinthevicinityofthedermis,whichislikelytobeongoingandrefractorytorapidhealing[6].AlthoughT-S/M-ShasdemonstratedvirucidalactivityandacapacitytoreduceviralloadinCElesions[18],thepenetrationofactivesintothedeeperbasalepitheliummaybeinsufficienttobecurative,enablingongoinginfectiontosustaintheSAAresponse.Multiple-doseregimensandlongertreatmentperiodsmaythereforebenecessary,potentiallyextendingforatleast4weeks,thetimeusuallyrequiredforlesionresolution.5.ConclusionsAlthoughT-S/M-ScannotbeexpectedtofullyeliminateORFVfromproliferativebasalepidermallesions,ourfindingssuggestthatitisapromisingtherapyforCE,capableofreducinginflammation,improvinganimalwelfareandhelpingcontrolsecondaryinfec-tionswithoutpromotingAMR.Further,ourresultsalsosupportthepotentialofSAAasabiomarkerformonitoringacutephaseresponsesduringCEandforassessingdiseasepro-gression.ThesefindingsshouldencouragefurtherresearchintooptimisingCEtreatmentprotocolsandtovalidateSAAasapracticaltoolformonitoringfieldoutbreaks.https://doi.org/10.3390/ani16010017

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Animals2026,16,17 10of12 AuthorContributions:Conceptualisation,P.A.W.,D.L.andA.O.;methodology,S.V.-S.,A.O.andD.L.;software,A.F.;validation,S.V.-S.andA.O.;formalanalysis,A.F.;investigation,P.Q.,H.R.,A.G.,D.G.andM.R.d.A.;resources,P.Q.,H.R.andM.R.d.A.;datacuration,A.F.;writing—originaldraftpreparation,A.O.;writing—reviewandediting,D.L.andP.A.W.;visualisation,A.F.andA.O.;supervision,D.L.;projectadministration,D.L.;fundingacquisition,P.A.W.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.Funding:ThisresearchwassupportedbyfundingandproductsfromtheAustraliancompanyMedicalEthics.ThisworkwasalsosupportedbytheAragónGovernment(A15_20RandA15_23R).InstitutionalReviewBoardStatement:AlltheproceduresweresupervisedandapprovedbytheEthicsAdvisoryCommissionforAnimalExperimentation(no.PI33/21),theBiosafetyCommitteeandtheOccupationalRiskPreventionUnitoftheUniversityofZaragoza,inaccordancewithcurrentregulationsregardingtheseprocedures,aspects:R.D.53/2013(whichalignswiththeEuropeanUnionDirective2010/63ontheprotectionofanimalsusedforexperimentalandotherscientificpurposes),Law31/1995,R.D.664/1997,R.D.1299/2006.InformedConsentStatement:Notapplicable.DataAvailabilityStatement:Therawdatasupportingtheconclusionsofthisarticlewillbemadeavailablebytheauthorsonrequest.Acknowledgments:TheauthorswouldliketoacknowledgetheinternsoftheRuminantClinicalServiceoftheVeterinaryFacultyofZaragozainvolvedinthisstudy.TheywouldalsoliketothankMariaÁngelesLostaoforhercollaborationinthelaboratorywork.ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest,althoughP.A.W.hasprovidedoccasionaladvisoryservicetoMedicalEthicsAustralia.Thefunderwasnotinvolvedinthestudydesign,collection,analysis,interpretationofdata,thewritingofthisarticleorthedecisiontosubmititforpublication.References1.Bala,J.A.;Balakrishnan,K.N.;Abdullah,A.A.;Mohamed,R.;Haron,A.W.;Jesse,F.F.A.;Nordin,M.M.;Mohd-Azmi,M.L.There-emergingoforfvirusinfection:Acallforsurveillance,vaccinationandeffectivecontrolmeasures.Microb.Pathog.2018,120,55–63.[CrossRef]2.Bergqvist,C.;Kurban,M.;Abbas,O.Orfvirusinfection.Rev.Med.Virol.2017,9,1932.[CrossRef]3.McElroy,M.C.;Bassett,H.F.Thedevelopmentoforallesionsinlambsnaturallyinfectedwithorfvirus.Vet.J.2007,174,663–664.[CrossRef]4.Nandi,S.;De,U.K.;Chowdhury,S.Currentstatusofcontagiousecthymaororfdiseaseingoatandsheep—Aglobalperspective.SmallRum.Res.2011,96,1366–1370.[CrossRef]5.Fleming,S.B.;Wise,L.M.;Mercer,A.A.MoleculargeneticanalysisofOrfVirus:Apoxvirusthathasadaptedtoskin.Viruses2015,7,1505–1539.[CrossRef][PubMed]6.Windsor,P.A.;Nampanya,S.;Tagger,A.;Keonam,K.;Gerasimova,M.;Putthana,V.;Bush,R.D.;Khounsy,S.Isorfinfectionarisktoexpandinggoatproductionindevelopingcountries?AstudyfromLaoPDR.SmallRum.Res.2017,154,123–128.[CrossRef]7.Rohde,J.;Emschermann,F.;Knittle,M.R.;Rziha,H.J.OrfvirusinterfereswithMHCclassIsurfaceexpressionbytargetingvesiculartransportandGolgi.BMCVet.Res.2012,8,114.[CrossRef][PubMed]8.Bukar,A.M.;Jesse,F.F.A.;Abdullah,C.A.C.;Noordin,M.M.;Lawan,Z.;Mangga,H.K.;Balakrishnnan,K.N.;Azmi,M.L.M.Immunomodulatorystrategiesforparapoxivirus:CurrentstatusandfutureapproachesforthedevelopmentofvaccinesagainstOrfvirusinfection.Vaccines2021,9,1341.[CrossRef][PubMed]9.Spyrou,V.;Valiakos,G.Orfvirusinfectioninsheeporgoats.Vet.Microbiol.2015,181,178–182.[CrossRef]10.Gumbrell,R.C.;McGregor,D.A.Outbreakofseverfatalorfinlambs.Vet.Rec.1997,141,150–151.[CrossRef]11.Hosamani,M.;Scagliarini,A.;Bhanuprakash,V.;McInnes,C.J.;Singh,R.K.Orf:Anupdateoncurrentresearchandfutureperspectives.Expert.Rev.Anti.Infect.Ther.2009,7,879–893.[CrossRef][PubMed]12.Lovatt,F.M.;Barker,W.J.W.;Brown,D.;Spooner,R.K.Case-controlstudyoforfinpreweanedlambsandanassessmentofthefinancialimpactofthedisease.Vet.Rec.2012,170,673.[CrossRef]13.Lawan,Z.;Bala,J.A.;Bukar,A.M.;Balakrishnan,K.N.;Mangga,H.K.;Abdullah,F.F.J.;Noor-din,M.M.;Mohd-Azmi,M.L.Contagiousecthyma:Howseriousisthediseaseworldwide?Anim.HealthRes.Rev.2021,22,40–55.[CrossRef]https://doi.org/10.3390/ani16010017

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Animals2026,16,17 11of12 14.Maor,D.;Yu,L.L.;Brand,R.Acaseoforfdiseaseinapatientwithscleroderma.JAADCaseRep.2017,3,155–157.[CrossRef]15.Kassa,T.AReviewonHumanOrf:ANeglectedViralZoonosis.Res.Rep.Trop.Med.2021,12,153–172.[CrossRef]16.Yeruham,I.;Perl,S.;Abraham,A.OrfInfectioninFourSheepFlocks.Vet.J.2000,160,74–76.[CrossRef]17.Lacasta,D.;Ferrer,L.M.;Ramos,J.J.;González,J.M.;Ortín,A.;Fthenakis,G.C.Vaccinationschedulesinsmallruminantsfarms.Vet.Microbiol.2015,181,34–36.[CrossRef]18.Lacasta,D.;Reina,R.;RuizdeArcaute,M.;Ferrer,L.M.;Benito,A.A.;Tejedor,M.T.;Echeverria,I.;Ruiz,H.;Cardenas,S.M.;Windsor,P.A.EffectofatopicalformulationoninfectiveviralloadinlambsnaturallyinfectedwithOrfvirus.Vet.Med.Res.Rep.2021,12,149–158.[CrossRef]19.Pye,D.Vaccinationofsheepwithcellculturegrownorfvirus.Aust.Vet.J.1990,67,182–186.[CrossRef][PubMed]20.Musser,J.M.B.;Taylor,C.A.;Guo,J.;Tizard,I.R.;Walker,J.W.Developmentofacontagiousecthymavaccineforgoats.Am.J.Vet.Res.2008,69,1366–1370.[CrossRef][PubMed]21.Zhao,K.;He,W.;Gao,W.;Lu,H.;Han,T.;Li,J.;Zhang,X.;Zhang,B.;Wang,G.;Su,G.;etal.OrfvirusDNAvaccinesexpressingORFV011andORFV059chimericproteinenhancesimmunogenicity.Virol.J.2011,8,562.[CrossRef]22.Yogisharadhya,R.;Bhanuprakash,V.;Kumar,A.;Mondal,M.;Schivachandra,S.B.ComparativesequenceandstructuralanalysisofIndianorfvirusesbasedonmajorenvelopeimmuno-dominantprotein(F1L),anhomologueofpoxviralp35/H3protein.Gene2018,663,72–82.[CrossRef][PubMed]23.Wassie,T.;Fammei,Z.;Jiang,X.;Liu,G.;Girmay,S.;Min,Z.;Chenhui,L.;Bo,D.D.;Ahmed,S.RecombinantB21andkisspeptin-54DNAvaccineinducesimmunityagainstorfvirusandinhibitsspermatogenesisinrats.Sci.Rep.2019,9,11.[CrossRef][PubMed]24.Wang,Y.;Sun,S.;Zhao,K.;Du,L.;Wang,X.;He,W.;Gao,F.;Song,D.;Guan,J.OrfvirusDNAprime-proteinbooststrategyissuperiortoadenovirus-basedvaccinationinmiceandsheep.Front.Immunol.2023,14,1077938.[CrossRef]25.Zhu,Z.;Qu,G.;Du,J.;Wang,C.;Chen,Y.;Shen,Z.;Zhou,Z.;Yin,C.;Chen,X.Constructionandcharacterizationofacontagiousecthymavirusdoublé-genedelectionstrainandevaluationofitspotentialasalive-attenuatedvaccineingoat.Front.Immunol.2022,13,961287.[CrossRef]26.Shen,Z.;Liu,B.;Zhu,Z.;Du,J.;Zhou,Z.;Pan,C.;Chen,Y.;Yin,C.;Luo,Y.;Li,H.;etal.Constructionofatriple-genedeletionmutantoforfvirusandevaluationofitssafety,immunogenicityandprotectiveefficacy.Vaccines2023,11,909.[CrossRef]27.Tontis,A.;Koning,H.;Kaderli,R.;Walter,L.Outbreaksofcontagiousecthymaintwoflocksofsheepandaherdofgoats.Schweiz.Arch.Tierheilkd.1981,123,19–28.28.Adedeji,A.;Adole,J.;Chima,N.;Maguda,A.;Dyek,D.;Jambol,A.;Anefu,E.;Shallmizhili,J.;Luka,P.ContagiousecthymainthreeflocksofgoatsinJos-southLGA,PlateauState,Nigeria.SokotoJ.Vet.Sci.2018,16,107.[CrossRef]29.Gómez,A.;Lacasta,D.;Tejedor,M.T.;RuizdeArcaute,M.;Ramos,J.J.;Ruiz,H.;Ortín,A.;Villanueva-Saz,S.;Reina,R.;Quilez,P.;etal.UseofalocalanaestheticandantisepticwoundformulationforthetreatmentoflambsnaturallyinfectedwithOrfvirus.Vet.Microbiol.2024,292,110037.[CrossRef]30.Greig,A.;Linklator,K.;Clark,W.Persistentorfinaram.Vet.Rec.1984,115,49.[CrossRef]31.Lacasta,D.;Ríos,M.;RuizdeArcaute,M.;Ortín,A.;Ramos,J.J.;Villanueva-Saz,S.;Tejedor,M.T.;Ruiz,H.;Borobia,M.;Reina,R.;etal.Useofalocalanaesthetic/antisepticformulationforthetreatmentoflambsexperimentallyinfectedwithOrfvirus.Animals2023,13,2962.[CrossRef][PubMed]32.Roberts,C.D.;Windsor,P.A.Innovativepainmanagementsolutionsinanimalsmayprovideimprovedwoundpainreductionduringdebridementinhumans:Anopinióninformedbyveterinaryliterature.Int.WoundJ.2019,16,968–973.[CrossRef][PubMed]33.Windsor,P.A.;Khounsy,S.;Earp,F.;MacPhillamy,I.;Young,J.;Bush,R.managingwelfareandantimicrobia-resistanceissuesintreatingfoot-and-mouthdiseaselesions:Anewtherapeuticapproach.Vet.Med.Res.Rep.2020,11,99–107.[CrossRef]34.Lendzele,S.S.;Mavoungou,J.F.;Burinyuy,K.A.;Armel,K.A.;Dickmu,S.J.;Young,J.R.;Thomson,P.C.;Windsor,P.A.EfficacyandapplicationofanoveltopicalanaestheticwoundformulationfortreatingcattlewithFoot-and-Mouthdisease:AfieldtrialinCameroon.Transbound.Emerg.Dis.2021,68,2531–2542.[CrossRef]35.Ceciliani,F.;Ceron,J.J.;Eckersall,P.D.;Sauerwein,H.Acutephaseproteinsinruminants.J.Proteomics2012,75,4207–4231.[CrossRef][PubMed]36.Petersen,H.H.;Nielsen,J.P.;Heegaard,P.M.H.Applicationofacutephaseproteinmeasurementsinveterinaryclinicalchemistry.Vet.Res.2004,35,163–187.[CrossRef]37.Eckersall,P.D.Proteins,proteomics,andthedysproteinemias.InClinicaBiochemistryofDomesticAnimals,6thed.;Kaneko,J.J.,Harvey,J.W.,Bruss,M.L.,Eds.;ElsevierAcademicPress:Davis,CA,USA,2008;pp.117–155,ISBN9780123704917.38.Tothova,C.;Nagy,O.;Kovac,G.Acutephaseproteinsandtheiruseinthediagnosisofdiseasesinruminants:Areview.Vet.Med.2014,59,163–180.[CrossRef]39.Saco,Y.;Bassols,A.Acutephaseproteinsincattleandswine:Areview.Vet.Clin.Pathol.2023,52,50–63.[CrossRef]40.Murata,H.;Shimada,N.;Yoshioka,M.Currentresearchonacutephaseproteinsinveterinarydiagnosis:Anoverview.Vet.J.2004,168,28–40.[CrossRef]https://doi.org/10.3390/ani16010017

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Animals2026,16,17 12of12 41.Reczy´nska,D.;Zalewska,M.;Czopowicz,M.;Kaba,J.;Zwierzchowski,L.;Bagnicka,E.Acutephaseproteinlevelsasanauxiliarytoolindiagnosingviraldiseasesinruminants:Areview.Viruses2018,10,502.[CrossRef]42.Navarro,T.;González,J.M.;Ramos,J.J.;Marca,M.C.;Figliola,L.;RuizdeArcaute,M.;Borobia,M.;Ortín,A.Impactofstressonhealthandfinalweightinfatteninglambs.Animals2020,10,1274.[CrossRef]43.Ferrer,L.M.;Lacasta,D.;Ortín,A.;Ramos,J.J.;Tejedor,M.T.;Borobia,M.;Pérez,M.;Castells,E.;RuizdeArcaute,M.;Ruiz,H.;etal.Impactodatopicalanaesthesiawoundmanagementformulationonpain,inflammationandreductionofsecondaryinfectionsaftertaildockinginlambs.Animals2020,10,1255.[CrossRef]44.Lepherd,M.L.;Canfield,P.F.;Hunt,G.B.;Bosward,K.L.Haematological,biochemicalandselectedacutephaseproteinreferenceintervalsforweanedfemaleMerinolambs.Aust.Vet.J.2009,87,5–11.[CrossRef]45.Dinler,C.;Tuna,G.E.;Ay,E.;Ulutas,B.;Voyvoda,H.y.;Ulutas,P.A.ReferenceintervalsforserumamyloidA,haptoglobin,ceruloplasmin,andfibrinogeninapparentlyhealthyneonatallambs.Vet.Clin.Path.2020,49,484–490.[CrossRef]Disclaimer/Publisher’sNote:Thestatements,opinionsanddatacontainedinallpublicationsaresolelythoseoftheindividualauthor(s)andcontributor(s)andnotofMDPIand/ortheeditor(s).MDPIand/ortheeditor(s)disclaimresponsibilityforanyinjurytopeopleorpropertyresultingfromanyideas,methods,instructionsorproductsreferredtointhecontent.https://doi.org/10.3390/ani16010017