Histophilus somni-associated syndromes in sheep from Southern Brazil

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

Veterinary

Microbiology

Histophilus

somni-associated

syndromes

in

sheep

from

Southern

Brazil

Selwyn

A.

Headley

_,

_Alfredo

_H.T.

_Pereira

_,

_Luciana

_C.

_Balbo

_,

_Giovana

_W.

_Di

_Santia

_,

Ana

P.F.R.L.

Bracarense

_,

_Luiz

_F.C.

_Cunha

_Filho

_,

_Jackson

_Schade

_,

_Werner

_Okano

_,

Priscilla

F.V.

Pereira

_,

_Fábio

_Morotti

_,

_Lucienne

_G.

_{Preto-Giordano}

_,

_Rogério

_A.

_Marcasso

_,

Alice

F.

Alfieri

_,

_Júlio

_A.N.

_Lisbôa

_,

_Amauri

_A.

_Alfieri

a_{UniversidadeEstadualdeLondrina,DepartamentodeMedicinaVeterináriaPreventiva,LaboratóriodePatologiaAnimal,Londrina,PR,}

Brazil

b_{UniversidadeEstadualdeLondrina,DepartamentodeMedicinaVeterináriaPreventiva,UnidadedeProcessamentodeTecidos,}

Londrina,PR,Brazil

c_{UniversidadeEstadualdeLondrina,DepartamentodeMedicinaVeterináriaPreventiva,LaboratórioMultiusuárioemSaúdeAnimal,}

BiologiaMolecular,Londrina,PR,Brazil

d_{UniversidadeNortedoParaná,FaculdadedeMedicinaVeterinária,Arapongas,PR,Brazil}

e_{UniversidadeEstadualdeLondrina,DepartamentodeClínicasVeterinárias,MedicinadeGrandesAnimais,Londrina,PR,Brazil}

f_{UniversidadeEstadualdeLondrina,DepartamentodeMedicinaVeterináriaPreventiva,Micologia,Londrina,PR,Brazil}

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Articlehistory:

Received29May2017 Accepted24December2017 Availableonline15March2018 AssociateEditor:MilianeSouza

Keywords: Histophilosis Moleculartesting Bronchopneumonia Myocarditis Thromboticmeningoencephalitis

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t

HistophilussomniisaGram-negativebacteriumthatisassociatedwithadiseasecomplex

(termedhistophilosis)thatcanproduceseveralclinicalsyndromespredominantlyin cat-tle,butalsoinsheep.HistophilosisiswelldescribedinNorthAmerica, Canada,andin someEuropeancountries.InBrazil,histophilosishasbeendescribedincattlewith respira-tory,reproductive,andsystemicdisease,withonlyonecasedescribedinsheep.Thisreport describestheoccurrenceofHistophilussomni-associateddiseaseinsheepfromSouthern Brazil. Eight sheep withdifferent clinicalmanifestationsfrom fivefarmswere investi-gated bya combinationof pathologicaland molecular diagnostic methodsto identify additionalcasesofhistophilosisinsheepfromBrazil.Theprincipalpathologicallesions werethromboticmeningoencephalitis,fibrinousbronchopneumonia,pulmonaryabscesses, andnecrotizingmyocarditis.ThemainclinicalsyndromesassociatedwithH.somniwere thromboticmeningoencephalitis(n=4),septicemia(n=4),bronchopneumonia(n=4),and myocarditis(n=3).H.somniDNAwasamplifiedfrommultipletissuesofallsheepwith clin-icalsyndromesofhistophilosis;sequencingconfirmedthePCRresults.Further,PCRassays todetectPasteurellamultocidaandMannheimiahaemolyticawerenegative.Thesefindings con-firmedtheparticipationofH.somniintheclinicalsyndromesinvestigatedduringthisstudy, andaddstothepreviousreportofhistophilosisinsheepfromBrazil.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:selwyn.headley@uel.br(S.A.Headley).

https://doi.org/10.1016/j.bjm.2017.12.008

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

592

Introduction

Histophilus somni (formerlyHaemophilus somnus) isa

Gram-negativebacteriumthatisassociatedwithadiseasecomplex (collectivelytermedhistophilosis) thatcan produceseveral clinicalsyndromesincludingthrombotic meningoencephali-tis(TME),pleuritis,polysynovitis,arthritis, bronchopneumo-nia,septicemia,myocarditis,otitismedia,infertility,abortion, andmastitisinaffectedruminants.1–4_{Thesesyndromeshave}

beenreferredtoastheH.somnidiseasecomplex,HSDC.5

HistophilosisortheHSDC,occurspredominantlyincattle withsporadicreportsofdiseaseoccurringinseveralspecies ofsmall ruminants.Thisbacteriumhasbeen isolatedfrom healthy goatsinMexico6 _{and Hungary}7 _{and insheepfrom}

Ethiopia.8 _{ReportsofH.somni associateddiseases insheep}

includeTME,9–11 _pneumonia,9 _{reproductivedisorders,}12_and

endometritis9,13_;spontaneous14_{andexperimentallyinduced}

epididymitis15 _{havealsobeen described.Moreover,H.somni}

wasassociatedwithpneumoniainbighornsheep.16

Descriptions of the HSDC in Brazil are incipient with abortions,17 _{respiratory impairment,}18 _{systemic disease,}19

andTME20_{beingdescribedincattlefromdifferent}

geograph-icalregions;webelievethathistophilosismightbeathreatto thelocalbeefcattleindustry.18However,theparticipationof

H.somniassociateddiseaseinsheepfromBrazilisrestricted

toasinglecasereportofaewewithendometritis.13

Conse-quently,toidentifyadditionalcasesofhistophilosisinsheep fromBrazil,thisstudyinvestigatedthepossibleassociationof

H.somniinsheepmortalityfromdifferentregionsofParaná

State,SouthernBrazil(Fig.1).

Materials

and

methods

Farmsaffected,clinicalhistory,autopsy,and

bacteriologicalidentification

Animals from this study were from different cities within the stateof Paraná, Southern Brazil.Sheep from all farms weresubmittedtotheLaboratoryofAnimalPathology,UEL forroutineautopsyanddiagnostics.Thegeographical loca-tionsofthefivefarmsaffected,thebiologicaldataofaffected sheep,andtheclinicaloutcomeareresumedatTable1.Three farms(A, B,andC)are innorthernParaná, andthe others inthenortheastern(D)and southeastern(E)regionsofthis State.Allfarmscontainedbreedsofsheeprearedprimarily formeatproduction,withthenumberofsheepvaryingfrom 20(FarmD)to1250(FarmE).SheepatFarmsA,C,andEwere routinelyimmunizedagainstclostridialdiseases;inaddition, those at Farm C were also immunized againstcontagious ectimaandfoot-rot.Sheepattheotherfarmswerenot rou-tinelyimmunizedagainstinfectiousdiseaseagents;allsheep weremaintainedpredominantlyongrasslandpastures;water was obtainedfrom streams or artesian wells within these farms.

Apathy(n=6) andrespiratorydiscomfort(n=4) werethe predominant clinical manifestations reportedly observed; sheep#3wasfounddeadwithoutanyclinicalmanifestation ofdisease.Acesareansectionwasdoneonsheep#4dueto

ruminalatony,abnormalgaitandapathy,anda3-monthold fetusthatdiedintra-uteruswassurgicallyremoved.However, theewediedspontaneouslytwodayslater.Inaddition,one neonatelamb(#1)hadomphalitis,enlargedkneeandelbow joints and locomotory difficulties. Theevolution ofclinical manifestationswas acute inall casesand all died sponta-neously.

A routineautopsywas done soonafter thedeath ofall animals; selected tissue fragments (cerebrum, cerebellum, lung,myocardium,liver,kidneys,andintestine)werefixedby immersion in10% bufferedformalinsolutionand routinely processedforhistopathologicalevaluation.Duplicatesections oftheorgansmentionedabovefromallanimals, aswellas sterileswabsfromtheaffectedjoints(kneeandelbow) and the umbilicalcordofanimal#1were collected freshly dur-ing necropsy, andmaintainedat−80◦_{Cuntil processedfor}

moleculartesting.Additionally,samplescollectedfreshlyat necropsywereplatedinBHIagarwith5%ofsheepbloodand processedformicrobiologicalcultureasdescribed.13

Extractionofnucleicacids,PCRassays,andsequencing

Nucleicacidsextractedfromtissuefragments(Table2)ofall animalsasdescribed,21_{wereusedinPCRassaysdesignedto}

amplifyspecificampliconsofbacterialpathogensassociated withsystemicdiseaseinruminants.Theseassaystargeting

the 16s rRNA gene ofH. somni,22 _{speciesand type-specific}

isolatesofPasteurellamultocida,23_{andthelktC-artJintergenic}

regionofMannheimiahaemolytica.24 _{Pulmonaryfragmentsof}

all animals were evaluated to identify the RNA of bovine respiratorysyncytialvirus,BRSV.25_{Purulentexudatefromthe}

abscesses of sheepwere collected freshly at necropsy and usedinaPCRassaydesignedtoidentifythe16SrDNAgene

ofTrueperella(Arcanobacterium) pyogenes(T.pyogenes).26

Posi-tivecontrolsincludednucleicacidsofH.somni,19_P.multocida,

M.haemolytica,andBRSVfrompreviousreports,27_{and from}

housekeepingsample(T.pyogenes).Nuclease-freewaterwas usedasnegativecontrolsinallPCRassays.PCRproductswere separatedbyelectrophoresisin2%agarosegels,stainedwith ethidiumbromide,andexaminedunderultravioletlight.The amplifiedPCRproductswerethenpurifiedandsubmittedfor direct sequencing using the DYEnamic ET Dye Terminator CycleSequencingKit(GEHealthcare,LittleChalfont,UK)with theforwardandreverseprimersinthe3500GeneticAnalyzer (AppliedBiosystems,Carlsbad,USA).

The obtained sequences were examined for quality analysis of chromatogram readings by using the PHRED software (http://asparagin.cenargen.embrapa.br/phph); sequences were only accepted if base qual-ity was equal to or greater than 20. Consensus sequences were then generated by the CAP3 program

(http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3.pl),

after which the partialnucleotide sequences were initially comparedbytheBasicLocalAlignmentSearchTool(BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST) with similar sequences deposited in GenBank. Phylogenetic tree and sequence alignments based on the 16s rRNA gene of the

Pasteurellaceae familywerethencreated byusingMEGA628_;

Model selectionindicatedthe Jukes-Cantormodel asbeing themostappropriatefordeterminationofthe phylogenetic

A

B

C

D

G

E

F

Fig.1–GrossdemonstrationofHistophilussomniassociatedlesionsinsheep.Thereismultifocalnecrotizingmyocarditis(A) andfibrinousbronchopneumoniawithabscess(B–C).Observetheseverelycongestedmeningealvessels(D),petechial epicardialhemorrhages(E),pulmonaryadhesions(F),andfibrinousbronchopneumoniawithpulmonaryabscess(G).Scale incentimeters.

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Table1–Geographicallocation,principalclinicalmanifestations,andoutcomeofsheepwithhistophilosis.

Sheep #

Farms Geographicallocation Biologicaldata Principalclinicalmanifestations Evolution(days) Outcome

1 A BomSucesso, NorthernParaná; n=200 Mortality,2 10-Day-old,lamb, Texel Apathy Dyspnea Locomotor difficulties Omphalitis Swollenjoints 2 Spontaneousdeath 2 B Londrina,Northern Paraná;n=30 Mortality,6 2-Year-old, Mixed-breed,ram Apathy Anorexia Bilateralnasal secretions Dyspnea Lateralrecumbency Nonproductive cough 1 Spontaneousdeath 3 3-Month-old, Mixed-breed,ewe

Founddead – Spontaneousdeath

4 C Londrina,Northern Paraná;n=65 Mortality,9 2-Year-old,Ilede France,ewe Abnormalgait Anorexia Apathy Congestedmucus membranes Recumbency Ruminalatony 3 Spontaneousdeath 5 D Cianorte, Northeastern Paraná;n=250 Mortality,20 2.5-Year-old, cross-breed,ewe Respiratory difficulties Bilateralseromucus nasalsecretion Diarrhea 4 Spontaneousdeath 6 E Ipiranga; Southeastern Paraná;n=1250; Mortality,150

12-Day-old,Texel Respiratory difficulties Apathy

12 Spontaneousdeath

7 12-Day-old,Texel Respiratory

difficulties Apathy

12 Spontaneousdeath

8 12-Day-old,Texel Respiratory

difficulties Apathy

12 Spontaneousdeath

relationship with the Maximum Likelihood method. The initialtreefortheheuristicsearchwasobtainedbyapplying the Neighbor-Joining method to a matrix of pairwise dis-tancesestimatedusingtheMaximumCompositeLikelihood approach.E.coliwasusedastheout-grouptoprovidestability tothegeneratedtree.

Results

Pathologicalfindingsandhistophilosissyndromes

TheprincipalpathologicalalterationsareresumedinTable2. Themostfrequentgrossfindings(Fig.2A–G)observedwere cranioventralpulmonaryconsolidation(n=6);congestionof meningeal vessels of the brain, petechial hemorrhages of the heart, and pulmonary edema were observed in four sheep.Multifocalmyocardialnecrosiswasobservedinsheep #2,whilepulmonaryabscesseswereobservedinsheep#5. Omphalitis(navel-ill)andmarkedswellingofbothkneeand elbowjoints (joint-ill) occurred in the neonatallamb (# 1).

Endometritiswassuspectedduetotheabnormaluterine exu-dateobservedinthesheep(#4)thathadadeadfetusremoved surgically.

The principal histopathological findings (Fig. 2A–F) observedinaffected sheepincludedTME(n=4),necrotizing myocarditis(n=4),purulent(n=2)andfibrinous(n=3) bron-chopneumonia, and pulmonary abscesses (n=1). Thelamb (# 1) withomphalitisand jointill hadTME, purulent bron-chopneumoniawithwidespreadthrombosisthataffectedthe kidneys,lungs,andlymphnodes.Further,TMEand necrotiz-ingmyocarditiswereobservedintheewe(#4),whilevascular congestion of the brain, lungs, and kidneys, suggestive of septicemia,occurredinher3-month-oldfetusthatsuffered intrauterinefetaldeath.

TheclinicalsyndromesassociatedwithH.somniobserved duringthisstudywere:(a)bronchopneumonia,withlesions insheep#5,6,7,and8;(b)myocardialdisease,observedin sheep#1,3,and4;(c),TME,affectingsheep#2,4,5,and8, (d)septicemia,sheep#1,3,4,and5;and(e)omphalitisand polyarthritisinsheep#1.

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 591–600

595

Grosslesions Histopathological

findings

Cerebrum Cerebellum Lung Heart Other

organs/tissues 1;10-day-old, lamb Congestionofmeningeal vessels Hydroperitoneum Omphalitis Petechiaeatthymus Polyarthritis Pulmonaryedema Pericardium:petechial hemorrhages Purulent bronchopneumonia Cerebralhemorrhageand thrombosis

Necrotizingmyocarditis Pulmonaryedema Thrombi(kidneys,lungs, lymphnodes,and cerebellum) Thymichemorrhage Myocarditis Omphalitis Polyarthritis Septicemia NC NC + + Kneejoint Umbilicalveina 2; Mixed-breed, ram Cranioventralpulmonary consolidation Hydropericardium Pulmonaryedema Multifocalmyocardial necrosis Serousatrophyof pericardialfat Brainstem: rhombencephalitisand thrombosis Cerebrum: meningoencephalitisand thrombosis Liver:hepatocelular degenerationand thrombosis Necrotizingmyocarditis Purulent bronchopneumonia TME +a ₊a _{− ND Brainstem} 3; Mixed-breed, ewe Cerebrum:congestionof meningealvessels Pulmonaryedema Cranioventralpulmonary consolidation Haemonchosis Monieziosis Pericardium:petechial hemorrhages Thrombotichepatitis Hemorrhagicmyocarditis Pulmonaryedemaand congestion Cerebralcongestion Myocarditis Septicemia + − + + ND 4;Ilede France,ewe

Abomasalulcerationsdue toHaemonchuscontortus

Cerebrum:congestionof meningealvessels Hydrothorax

Kidneys:chocolatecolored Liver:increasedconsistency Myocardialhemorrhages Pulmonaryedema Uterus:emaciatedwith dark-reddishexudate

Necrotizingmyocarditis Cerebrum:

meningoencephalitis, bacterialembolism,and thrombosis

Chronichepatitiswith bridgingfibrosis Tubularrenalnecrosis Pulmonaryedema Myocarditis Septicemia TME +a _{+ + + Brainstem} Livera Kidney Spleen Uterus

596

Sheep# Pathologicalalterations Histophilosis

syndromes

H.somniDNA

Grosslesions Histopathological

findings

Cerebrum Cerebellum Lung Heart Other

organs/tissues 5;

Cross-breed, ewe

Abomasalulcerationsdue toHaemonchuscontortus

Adherence(pulmonary pleuralandribs) Cranioventralpulmonary consolidation

Hydropericardium Meninges:thickenedwith congestedvessels Pericardium:petechial hemorrhages Pulmonaryabscess Purulentnasalsecretion

Cerebrumandcerebellum: meningoencephalitiswith hemorrhage,thrombosis, andbacterialembolism Chronicproliferative hepatitis

Fibrinous

bronchopneumonia, thrombosis,andbacterial embolism

Hemorrhagicnephritisand thrombosis Necrotizingmyocarditis andthrombosis Liver:thrombosis Bronchopneumonia Septicemia TME +a _{ND + ND Pulmonaryabscess} 6;12-day-old, Texel Cranioventralpulmonary consolidation Fibrinous bronchopneumonia Bronchopneumonia ND ND + + ND 7;12-day-old, Texel Cranioventralpulmonary consolidation Fibrinous bronchopneumonia Bronchopneumonia ND ND + ND ND 8;12-day-old, Texel Cranioventralpulmonary consolidation Pleuritis Fibrinous bronchopneumonia Thrombotic meningoencephalitis Bronchopneumonia TME + ND + + ND

+,positive;−,negative;TME,thromboticmeningoencephalitis;ND,notdetermined;NC,notcollected.

A

D

B

E

C

F

Fig.2–HistopathologicalfindingsassociatedwithHistophilussomniinsheep.Thereisnecrotizingmyocarditis(A), bronchopneumonia(B),bacterialemboliwithinthebrain(C)andlung(D),andthrombosisatthecerebrum(E)andchoroid plexus(F).Bar:A,C-E,0.02mm;BandF,0.1mm.

Furthermore,repeatedattempts atbacterialcultureand identificationwerefrustrating,sincethegrowthofcolonies

ofH.somnididnotoccurfromanyofthesamplescollected.

MolecularidentificationofH.somniDNAintissues

H.somniDNAwasamplifiedfrommultipleaffectedtissuesof

allsheep(Table2).Further,thedisseminateddistributionof

H.somniDNAwasmorewidespreadinsheep#1,2,3,4,and

5.Inaddition,H.somniDNAwasamplifiedfromtheumbilical veinandjoints(kneeandelbow) oftheneonatallamb(#1) thathadnavelandjointill.ItmustbehighlightedthatH.somni

DNAwasidentifiedinmultipletissues(cerebrum,cerebellum, brainstem,liver,kidney,andspleen)ofthesheep#4aswellas inthekidneyandspleenofher3-month-oldfetus.Further,T.

pyogenesDNAwasamplifiedfromthepurulentexudateofthe

pulmonaryabscessofsheep#5,whereH.somniDNAwasalso identifiedbyPCRindicatingthatbothpathogensparticipated inthedevelopmentofthelungabscessofthissheep;allother PCRassaysyieldednegativeresults.

Partialsequencesofthe16srRNAgeneofH.somniwere obtained from the umbilical vein (GenBank accession No. KP419695)ofsheep#1,thecerebrumofsheep#2(KU726866), 6(KU726864),and5(KU726867),theliver(KU726865)ofsheep #4,andthekidney(KU726862)andspleen(KU726863)ofthe 3-month-old-fetus.InitialBLASTanalysesrevealedthatthese sequences demonstrated 98–99% identity with similar iso-latesofH.somnidepositedinGenBank.Phylogeneticanalyses revealed that the sequences derived from this study clus-tered withotherisolatesofH.somni(Fig.3);thenucleotide sequencesusedforphylogeneticanalysesduringthisstudy are givenin Fig. 3. Furthermore,the isolates derivedfrom

598

KU726863 Sheep Brazil KU726862 Sheep Brazil KU726864 Sheep Brazil KU726865 Sheep Brazil KU726866 Sheep Brazil

KU726867 Sheep Brazil KF770464 Brazil KF770474 Brazil KF770461 Brazil KF770472 Brazil KF770468 Brazil KF770467 Brazil KF770466 Brazil KF770465 Brazil KF177393 Brazil KF770462 Brazil KR185350 Sheep Brazil KR185349 Sheep Brazil KF770471 Brazil KF770470 Brazil K770469 Brazil KF770473 Brazil KF770463 Brazil KM374593 Brazil KM374593 Brazil KM374591 Brazil KM374590 Brazil KM374589 Brazil M35018 Pasteurella multocida M75068 Actinobacillus lignieresii AF060699 Mannheimia haemolytica

J01695. coli AF549389 Switzerland KP419695 Sheep Brazil NR074278 USA KC414854 Brazil KC414855 Brazil AF549388 USA AB176892 USA AB176891 USA AB176893 Japan AB176900 USA AF549403 Denmark AF549394 Australia AF549393 Canada AF549399 USA KF177392 Brazil KF770475 Brazil AF549398 Sheep USA AF549395 Sheep Australia AF549395 Sheep Canada AB176920 Sheep Canada AB176921 Sheep Canada

100

43

60

0.02

Fig.3–Phylogenetictreebasedonthe16SrRNAgene sequencesofHistophilussomniandselectedPasteurellaceae

membersgeneratedbyMEGA6.Thetreewasconstructed bytheNeighbor-Joiningmethod,basedon1000

bootstrappeddatasets;distancesvalueswerecalculatedby usingtheJukes-Cantorparametermodel.TheGenBank accessionnumbersandcountryoforiginofthesequences aregiven;thesequencesderivedfromthisstudyare highlighted(blackdot).

thecerebrumandliverofsheep#5wereidenticaltothose obtainedfromspleenandkidneyofher3-month-oldfetusthat sufferedintrauterinedeath.

Discussion

Theimportanceofthis reportliesistheidentification ofH.

somniDNAinmultipletissuesofsheepwith

histopathologi-callesionsthatcharacterizeseveraltypicalclinicalsyndromes ofHSDCpreviouslydescribedincattle,2–5_andsheep.9_These

resultsaddtotheoccurrenceofthispathogenasan impor-tantdiseaseagentofruminantsinthiscountry,considering the recent descriptions of H. somni-associated diseases,19

abortions,17 _{andTMEincattle,}20 _{aswell asendometritisin}

onesheep.13_{Consequently,thesefindingsrepresentthefirst}

studytoeffectivelyconfirmseveralclinicalsyndromes

associ-atedwithH.somniinsheepfromBrazil;apreviousstudyonly

identifiedthispathogeninoneewe.13_{Collectively,these}

find-ingscontributetothehypothesisthathistophilosisisindeed a threatnotonlyto beefcattlefrom Brazil,18 _{but to}

rumi-nantsonawiderscale,principallyduetothedevelopmentof respiratory,reproductive,andneurologicaldiseasein suscep-tibleherds.Further,wepostulatethatcasesofhistophilosis inBrazilareprobablyunderdiagnosedsincemostveterinary cliniciansmighthaveconfusedthesesyndromeswithother diseaseprocessesofruminants.

The results from this study have identified the DNA

of H. somni within multiple tissues as well as from the

kneejointandaffectedumbilicalcordoftheneonatallamb (#1),therebycharacterizingsystemichistophilosis.Moreover, nucleotidesequencingoftheampliconsconfirmedthese find-ings, associating this bacterium with the pathogenesis of theomphalitis(navel-ill),arthritis(joint-ill), bronchopneumo-nia,andmyocarditishereindescribed.Inaddition,thissheep alsopresentedTMEandmyocarditis,clinicalsyndromes fre-quentlyassociatedwithH.somniincattle,29–31_andsheep9,10_;

thesesyndromeswerealsodemonstratedexperimentallyin sheep.32

Another interesting finding during this study was the amplificationofH.somniDNAfrommultipletissuesofsheep # 5 and her fetus, confirming the vertical transmissionof this pathogen;similar results were described in dairy cat-tlefromdifferentgeographicalregionsofBrazil.17_Although

thiswasnotanabortionperse,infectionbyH.somniresulted infetaldeath,suggestingthatthispathogen isalso associ-atedwithreproductivedisordersinsheep,sinceH.somniDNA wasidentifiedinramswithepididymitis,9,14_{andineweswith}

endometritis.9,13 _{Alternatively,H.somniisaknown}

abortifa-cientagentofcattlewithdescriptionsofnaturalinfectionsin countriesincludingCanada,33_theUK,34_USA,35_andBrazil.17

Thephylogeneticanalysisdemonstratedthattheisolates

ofH.somniderivedfrom thisstudy clusteredwiththoseof

cattleand sheepfrom differentgeographicallocations;this suggest theclosephylogeneticrelationshipofthesestrains irrespectiveoftheirgeographicalorigin,confirmingthatthe same pathogen affects cattleand sheep,aswaspreviously demonstrated.36_{Unfortunately,bacterialisolationwouldhave}

been idealto definethe biochemical characteristics ofthis pathogenbutgrowthofH.somniwasnotsuccessful.Cultureof

H.somnifrominfectedtissuesisconsideredadifficulttask,4,22

whichmayexplainthefrustratedattemptsatcultureduring thisinvestigation.Inthisregard,PCRidentificationwith sub-sequentsequencing,ashereindescribed,isrecommendedto identifythisbacterialpathogenfrominfectedtissuesor envi-ronmentalsamples.22

Conclusion

H.somniDNAwasidentifiedinmultipletissuesofsheepfrom

different geographical regions of the state of Paraná with clinicalsyndromesofhistophilosisconfirmedby histopatho-logicalevaluations.Thesefindingssuggestthatthisbacterial pathogenwasassociatedwithsheepmorbidityandmortality fromtheseregions.

Conflicts

of

interest

Theauthorsdeclarethattheyhavenoconflictsofinterest.

Acknowledgements

S.A.Headley,A.P.F.R.L.Bracarense.,A.F.Alfieri,J.A.N. Lisboa, and A.A. Alfieri are recipients of the National Coun-cil for Scientific and Technological Development (CNPq; Brazil) fellowships and grants. This study was partially funded by a CNPq grant (Proposal #448797/2014-3) and a joint CNPq/Ministry of Agriculture (MAPA) grant (Protocols #578645/2008-4and#478254/2012-1).

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