Pathological, Bacteriological and Virological Findings in Sudden and Unexpected Deaths in Young Dogs

Abstract

In human medicine, “sudden death” has been defined by the World Health Organization (WHO) as a non‐violent, unexpected death occurring less than 24 h from the onset of symptoms. The aims of this study were: (1) to estimate the proportional mortality ratio for “sudden and unexpected death” (SUD) in young dogs; (2) to investigate the pathological and microbiological findings in SUD cases in young dogs. For these purposes, a retrospective study of a total of 145 cases of young dead dogs was performed. For each case, we collected information about the age, medical history and the gross and microbiological findings of the animals. The results of this study found 21 cases of SUD. The most frequently observed clinical symptoms in the cases of sudden death were acute respiratory symptoms, followed by acute gastroenteric symptoms, non‐specific symptoms and neurological symptoms. The evaluation of necropsy reports allowed us to observe enteritis in 18 out of 21 cases and pneumonia in seven out of 21 cases. Viral infection with Canine parvovirus type 2 was the most common cause of SUD observed. These results could provide a valuable tool for the investigation of sudden death in young dogs.

Full text

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Animals2020,10,1134;doi:10.3390/ani10071134www.mdpi.com/journal/animals
Article
Pathological,BacteriologicalandVirological
FindingsinSuddenandUnexpectedDeathsin
YoungDogs
GiuseppePiegari
1,
*,LorenaCardillo
2
,FloraAlfano
2
,LuciaVangone
2
,ValentinaIovane
3

andGiovannaFusco
2

1
DepartmentofVeterinaryMedicineandAnimalProduction,UnitofPathology,UniversityofNaples
FedericoII,Naples80137,Italy
2
IstitutoZooprofilatticoSperimentaledelMezzogiorno,Portici,81100Naples,Italy;
lorena.cardillo85@gmail.com(L.C.);flora.alfano@cert.izsmportici.it(F.A.);
lucia.vangone@izsmportici.it(L.V.);giovanna.fusco@izsmportici.it(G.F.)
3
DepartmentofPharmacy,UniversityofSalerno,84084Fisciano,Italy;viovane@unisa.it
*Correspondence:giuseppe.piegari@unina.it
Received:6May2020;Accepted:2July2020;Published:3July2020
SimpleSummary:“Suddendeath”hasbeendefinedbytheWorldHealthOrganizationasanon‐
violent,unexpecteddeathoccurringlessthan24hfromtheonsetofsymptoms.Thecausesof
suddendeathhavebeenwidelyinvestigatedinhumanforensicmedicine.Incontrast,fewstudies
havebeenreportedintheveterinaryliterature.Thisstudyaimedtoinvestigatethefrequencyof
suddendeathsinyoungdogsindifferentageranges.Asecondaryaimwastocollectinformation
regardingclinicalsymptoms,andpathologicalandmicrobiologicalfindingsrelatedtosuddendeath
inyoungdogs.Theresultsofthepresentstudydemonstratethatthehighestfrequencyofsudden
deathoccursinanimalsinanagerangefrom10daysto1monthandfrom6to12months.Themost
frequentlyobservedclinicalsymptomsincasesofsuddendeathwereacuterespiratorysymptoms.
Furthermore,Canineparvovirustype2,E.coli,CanineDistemperVirus,ClostridiumperfringenstypeA,
andPasteurellaspp.werethemaincausesofdeathobservedinthepresentstudy.Theresults
reportedinthepresentstudycouldprovideareferencebasistobetterinvestigatesuddendeathin
veterinaryclinicalpractice.
Abstract:Inhumanmedicine,“suddendeath”hasbeendefinedbytheWorldHealthOrganization
(WHO)asanon‐violent,unexpecteddeathoccurringlessthan24hfromtheonsetofsymptoms.
Theaimsofthisstudywere:(1)toestimatetheproportionalmortalityratiofor“suddenand
unexpecteddeath”(SUD)inyoungdogs;(2)toinvestigatethepathologicalandmicrobiological
findingsinSUDcasesinyoungdogs.Forthesepurposes,aretrospectivestudyofatotalof145cases
ofyoungdeaddogswasperformed.Foreachcase,wecollectedinformationabouttheage,medical
historyandthegrossandmicrobiologicalfindingsoftheanimals.Theresultsofthisstudyfound21
casesofSUD.Themostfrequentlyobservedclinicalsymptomsinthecasesofsuddendeathwere
acuterespiratorysymptoms,followedbyacutegastroentericsymptoms,non‐specificsymptoms
andneurologicalsymptoms.Theevaluationofnecropsyreportsallowedustoobserveenteritisin
18outof21casesandpneumoniainsevenoutof21cases.ViralinfectionwithCanineparvovirustype
2wasthemostcommoncauseofSUDobserved.Theseresultscouldprovideavaluabletoolforthe
investigationofsuddendeathinyoungdogs.
Keywords:post‐mortemmicrobiology;veterinaryforensicpathology;suddendeath;youngdogs
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Animals2020,10,11342of12
1.Introduction
Inhumanmedicine,“suddendeath”hasbeendefinedbytheWorldHealthOrganization
(WHO)asanon‐violent,unexpecteddeathoccurringlessthan24hfromtheonsetofsymptoms[1];
inparticular,theterm“suddenandunexpectedinfantdeath”(SUID)isusedtodescribedeathsthat
occurrelativelysuddenlyandunexpectedlyinchildrenlessthan1yearsold[2,3].Infectionsare
reportedintheliteratureasanimportantcauseofSUID,followedbymetabolicormolecular
disorders[2–5].ThemainpathogensreportedinSUIDcasesareasfollows:Staphylococcusaureus,
Escherichiacoli,Streptococcuspyogenes,Streptococcusagalactiae,Streptococcuspneumoniae,GroupB
Streptococci(GBS),RespiratorySyncytialVirus(RSV),Cytomegalovirus(CMV)andAdenovirus[2,6,7].
However,abroadrangeofpathogenshasbeenreportedintheliteratureasbeingcausesoraco‐
factorsinSUID,suchasParvovirusB19,Epstein‐Barrvirus,InfluenzaAvirusandMycobacterium
tuberculosis[2,6,8,9].Furthermore,recentstudiesreportedtherelativelybenignCoxsackievirusA16
asapossiblecontributingfactorinSUIDinhumans[10].Forthesereasons,thecurrentSUIDautopsy
protocolintheUKandtheinternationalguidelinesadvocateforamultidisciplinaryapproachtothe
investigationofallcasesofSUID,whichshouldbebasednotonlyonthefindingsofthemacroscopic
examination,butalsoonabroadrangeofancillaryinvestigations,suchasbacteriologicaland
virologicalanalyses[11,12].Suddeninfantdeathsyndrome(SIDS)isconsideredtobeasub‐classof
SUID,inwhichthecauseofdeathremainsunexplainedevenaftertheforensicnecropsy,ancillary
testsandevaluationoftheanamnesticdataandcrimesceneanalysis[1,2].Indeed,amongthecases
ofSUID,only20%haveaclearcause,whilemostcasesremainunexplainedandarecategorizedas
SIDS[1,5,13].Althoughthecauseisunknown,specificgeneticmutationsormildinfectionscouldbe
involvedinthegenesisofthesyndrome[5].Mildinfectionshavebeensuggestedtoplayakeyrole,
asdemonstratedbyalteredlevelsofimmunoglobulinorcytokineandthehighfrequencyofmild
trachealinfectionscommonlyobservedduringpost‐mortemexaminationsofthesubjectswithafinal
diagnosisofSIDS[5].Although,inhumanmedicine,theconceptofsuddendeath,SUIDandSIDS
hasbeenwelldefinedbytheWHO,inveterinarymedicine,auniversaldefinitionislacking.Some
authorshavedefinedsuddendeathinanimalsasdeaththatoccursinafewminutesorseveralhours,
duetopre‐existingdiseaseorafunctionaldisorder[14].However,intheopinionoftheauthors,this
definitionshouldbeavoided,becauseitlacksawell‐definedtemporalreferencerange.Incontrast,
evenifnotyetvalidatedinveterinarymedicine,theWHOdefinitionprovidesanimportanttemporal
referencerangeusefulfortheidentificationofcasesofsuddendeathinveterinaryclinicalpractice.
Overthelastfewyears,manystudieshaveinvestigatedthecauseofdeathinanimals.In
particular,infectiousdiseasesthataffectthegastrointestinalsystemarereportedtobethemaincause
ofdeathinpuppiesandyoungdogs[15,16].Incontrast,neoplasticdiseasesappeartobetheprevalent
causeofdeathinadultdogs[15].Amongtheinfections,canineparvovirustype2(CPV‐2)isreported
tobeoneofthemostcommonandimportantcausesofmorbidityandmortalityinyoungdogs[17,18].
Moreover,thisvirusisconsideredtobeanimportantpathogenresponsibleforacutegastroenteritis
andmyocarditisindogs[17,18].However,withregardstosuddenandunexpecteddeaths,despite
theunderlyingcauseshavingbeensporadicallyinvestigatedindogs[19],tothebestofour
knowledge,nostudyhasevaluatedthemicrobiologicalfindingsincasesofsuddendeathinyoung
dogs.Inlightoftheseobservations,theaimsofthisstudywereasfollows:(1)toestimatethe
proportionalmortalityratio(PMR)for“suddenandunexpecteddeath”inpuppiesandyoungdogs;
(2)toinvestigatethepathological,bacteriological,andvirologicalfindingsinsuddenandunexpected
deathinyoungdogs;and(3)tointroduceastandardizedmicrobiologicalprotocolforthediagnostic
investigationofcasesofsuddendeathinveterinarymedicine
2.MaterialsandMethods
2.1StudyDesign
Anobservationalretrospectivestudyofatotalof145casesofyoungdeaddogs,consecutively
presentedbyveterinarypractitioners,owners,orlawenforcementtothe“IstitutoZooprofilatticodel
Mezzogiorno”(IZSM)ofPorticicity,SouthernItaly,wasperformedovera3‐yearperiod(2015–2017).

Animals2020,10,11343of12
Thesubmissionformswerecollectedtoobtaininformationaboutthemedicalhistoryandageofthe
animals.Onthebasisofthemedicalhistory,theanimalsweredividedintogroupsasfollows:
‐ (Suddenandunexpecteddeathgroup—SUDgroup):dogswithaclinicaldiagnosisofsudden
andunexpecteddeath.AccordingtotheWHO,suddenandunexpecteddeath(SUD)caseswere
consideredtobeanon‐violentandunexpecteddeaththatoccurslessthan24haftertheonsetof
symptoms;
‐ (Expecteddeathgroup—EDgroup):dogswithoutaclinicalhistoryofsuddenandunexpected
death
Onthebasisofage,theavailabledatawerecategorizedasfollows:(Group1)10days–4weeks;
(Group2)4weeks–6weeks;(Group3)6weeks–2months;(Group4)2–3months;(Group5)3–6
months;(Group6)6–12months.Eachexaminedcasewassubjectedtoacompletenecropsyand
bacteriologicalandvirologicalinvestigations;however,forthepurposesofthisstudy,onlythe
necropsyandmicrobiologicalreportsfromanimalsintheSUDgroupwereincluded.Furthermore,
themicrobiologicalinvestigationswererestrictedtomoleculartestsforthevirologicalanalysis,and
microbiologicalculturesforthebacteriologicalexaminations.Inallassessedcases,moleculartests
wereperformedusingareal‐timepolymerasechainreactionassay(RT‐PCR)forcanineparvovirus
(CPV),caninecoronavirus,canineadenovirus,herpesvirus,andcaninedistempervirus.Furthermore,inall
casespositiveforcanineparvovirustype2,multiplexedPCRpanelswereusedtodistinguishbetween
wild‐typeandvaccineCPV‐2andtoidentifythepathogensubtype(CPV‐2a;CPV2b;CPV2c)[20].
Theanalyzedsamplesincludedtheliver,lung,kidney,spleen,heart,brainandintestine.
MicrobiologicalresultsandnecropsyreportswerebothextractedfromtheIZSMinformationsystem
(SIGLA).Animalsthattestedpositiveinthetoxicologicalinvestigations,orthathaddiedfrom
trauma,wereexcludedfromthestudy.
2.2.NecropsyProtocol
Allnecropsieswereperformedinthenecropsyroomofthe“IstitutoZooprofilattico
SperimentaledelMezzogiorno”(IZSM),Portici,Italy,withastandardnecropsyprotocol[21].All
SUDcaseswerestoredat4°Cbeforenecropsy.Theperiodbetweendeathandnecropsywasbetween
12and36h.Duringthenecropsy,accordingtointernalinstituteprotocol,allsamplesweretakenin
rigorousasepsisconditionsusingsterileinstrumentsandtransportedtothelaboratoryof
microbiology.Furthermore,toobtainuncontaminatedspecimens,asterilizationofthebodyand
organssurfaceswasperformedbeforesampling.Finally,themeantimebetweensamplecollection
andtransporttothereferencelaboratorywasunder3h.
2.3.AnalyticalValidationoftheResults
Foreachcaseofsuddenandunexpecteddeath,theclinicalhistory,necropsyreportand
microbiologicalfindingswerereviewed,andthefinalcauseofdeathwascategorizedas“explained”
or“unexplained”.However,sincedeterminingthepathologicalsignificanceofthemicroorganisms
isolatedduringnecropsyisoftendifficult,ashasbeenfrequentlyreportedintheliterature[22,23].
Forthepurposesofthisstudy,virusesdetectedbyPCRwereconsideredtobethecauseofdeath,
onlywhenassociatedwiththetypicalmacroscopicchangesobservedduringthe
anatomopathologicalexamination.Inaddition,thebacteriologicalandvirologicalfindingswere
interpretedconsideringabroadrangeofvariables,suchasthelocationofpathogendetection,the
capacityofpathogensforvirulence,thecorrelationwithinjuriesobservedduringthenecropsy,the
multisitelocationofthepathogens,theageofthedogandthecompositionofthenormalflora.
2.4.StatisticalAnalysis
Thefrequenciesofsuddenandunexpecteddeath(SUD),expecteddeath(ED),andtotaldeaths
(SUD+ED)wereevaluatedandstratifiedbyageclasses.Furthermore,weestimatedtheproportional
mortalityratio(PMR)for“SUD”ineachassessedagegroup.TheChi‐squaretestwasusedtoassess
differencesinthedistributionsofEDandSUDamongagegroups.

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3.Results
Outofthe145examinedreports,wefound21casesofSUDand124casesofEDduringthe3‐
yearstudyperiod.ThePMRofSUDwastherefore14.48%,whiletheEDwas85.52%.Furthermore,
theChi‐squaretestshowedasignificantdifferenceinthefrequenciesofEDandSUDamongthe
assessedagegroups(p<0.05).AllSUDcasesweresubmittedbyItalianveterinarypractitioners.The
highestfrequenciesofexpecteddeathwereobservedinanimalsinGroup2(100%ofthecases),Group
3(87.7%vs.12.5%),Group4(97.2%vs.12.8%)andGroup5(93.3%vs.6.7%).Incontrast,thehighest
frequenciesofSUDwerefoundinanimalsinGroup1(58.8%vs.41.2%)andGroup6(37.5%vs.
62.5%).Table1summarizesthefrequenciesandpercentagesofSUDandEDandthefrequencyof
totaldeaths(SUD+ED)stratifiedbyageclasses.Overall,ofthe21SUDcases,10outof21(47.61%)
dogswerelessthan4weeksold(Group1),0outof21werebetween4weeksand6weeksold(Group
2),fiveoutof21(23.8%)werebetween6weeksand2monthsold(Group3),oneoutof21(4.76%)
wasbetween2and3monthsold(Group4),twooutof21(9.51%)werebetween3and6monthsold
(Group5),andthreeoutof21(14.28%)werebetween6monthsand1yearold(Group6).
Table1.Frequencyandpercentageofsuddenandunexpecteddeaths,expecteddeathsandthe
frequencyoftotaldeathsstratifiedbyagegroups.
AgeGroupSuddenDeathExpectedDeathTotalDeaths
Group110(58.8%)7(41.2%)17
Group2014(100%)14
Group35(12.5%)35(87.5%)40
Group41(2.8%)35(97.2%)36
Group52(6.7%)28(93.3%)30
Group63(37.5%)5(62.5%)8
Total21124145
3.1ClinicalBackgroundandGrossFindings
Themostfrequentlyobservedclinicalsymptomsincasesofsuddendeathwereasfollows:acute
respiratorysymptomsin12outof21cases,followedbyacutegastroentericsymptoms(asingleor
fewepisodesofvomitingordiarrhea)insixoutof21cases,neurologicalsymptomsinonecase,and
finally,non‐specificsymptomsintwooutof21cases.Theevaluationofnecropsyreportsallowedus
toobservehaemorrhagicgastroenteritisin12outof21cases,pneumoniainsevenoutof21cases,and
catarrhalenteritisinfiveoutof21cases(Figure1).Pulmonaryoedemaormultiorgancongestion
werealsoobservedin19outof21cases.

Figure1.Pathologicallesionsincasesofsuddendeath.(A):pneumonia(arrows)(B):haemorrhagic
gastroenteritis(C):catarrhalenteritis.
3.2.MicrobiologicalExamination
Inallanimalsdeadforsuddenandunexpecteddeath,virologicalinvestigationswereperformed
withapanelofvirusestestedbyPCR(Canineparvovirus,Caninecoronavirus,Canineadenovirus,Canine
herpesvirusandCaninedistempervirus),andabacteriologicalexaminationwasperformedwith

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microbiologicalcultures.Theretrospectiveanalysisshowedpositivemicrobiologicalresultsin18out
of21cases(Table2).
Table2.Virusesorbacteriadetectedincasesofsuddenandunexpecteddeath.
PathogenNO.ofCases
CanineParvovirus10
E.Coli7
ClostridiumperfringenstypeA6
Adenovirus3
CanineDistemperVirus2
Streptococcussanguinis2
Pasteurellaspp.2
Streptococcusdysgalactiae1
Rotavirus1
However,forthepurposesofthisstudy,themicrobiologicalfindingswereinterpreted
consideringabroadrangeofvariables.Inparticular,thelocationofpathogendetection,thecapacity
ofpathogensforvirulenceandthecorrelationofthatmicroorganismswiththeobservedmacroscopic
injurieswerethemostimportantparametersassessedinthisstudy.Therefore,afterthereviewofthe
necropsiesandmicrobiologicalreports,thedetectedpathogenswereconsideredthemaincauseof
deathinonly14outof21cases.Inparticular,amongtheevaluatedcases,themaincauseofdeath
wasviralinfectionwithCanineparvovirustype2(8/21),followedbyviralinfectionwithCanine
parvovirustype2,andco‐infectionwithE.coli(2/21),bacterialco‐infectionwithClostridiumperfringens
typeAandE.coli(2/21)andviralandbacterialco‐infectionwiththeCaninedistempervirusand
Pasteurellaspp.(2/21).Finally,insevenoutof21cases,themicrobiologicalresultsdidnotexplainthe
injuriesobservedduringthenecropsy.Therefore,thecausativeagentofinfectionwasconsidered
undeterminedafterthemicrobiologicalexamination.Table3summarizestheclinicalbackgrounds,
pathologicalfindings,microbiologicalresultsandcausesofdeathofthecasesofsuddendeath.

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Table3.Microbiologicalandanatomopathologicalfindingsofthestudieddogs.
GroupSexClinicalBackgroundPathologicalFindingsVirological
ExaminationBacteriologicalExaminationCauseofDeath
1
MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,
hemorrhagicgastroenteritis
Canineparvovirustype2a
(wildtype)—detectedin
thelung,liver,heart,
brain,andintestine
Streptococcusdysgalactiae—
isolatedinthelungViralinfection
MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,severe
broncho‐pneumonia,
catarrhalenteritis
NovirusesdetectedStreptococcussanguinis—
isolatedintheintestine
Undetermined:
severepneumoniadue
tounexplainedcauses
MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,severe
broncho‐pneumonia,
catarrhalenteritis
NovirusesdetectedStreptococcussanguinis—
isolatedintheintestine
Undetermined:
severepneumoniadue
tounexplainedcauses
FAcuterespiratory
insufficiency
Visceralcongestion,
multifocalpulmonary
hemorrhages,hemorrhagic
enteritis
Canineparvovirustype2b
and2c(wildtype)—
detectedinthelung,
liver,spleen,heart,and
intestine
Clostridiumperfringens,
DetectionofClostridium
perfringensalphatoxin—
isolatedintheintestine
Viralinfection
MAcuterespiratory
insufficiency
Lobarpneumonia,
pulmonaryedema,
catarrhalenteritis
Caninedistempervirus—
detectedinthelung,
liver,andbrain
Pasteurellaspp.—detectedin
thelungViralandbacterialinfection
FAcuterespiratory
insufficiency
Visceralcongestion,
multifocalpulmonary
hemorrhages,hemorrhagic
enteritis
Canineparvovirustype2b
and2c(wildtype)—
detectedinthelung,
liver,brain,heart,and
intestine
E.coli,Clostridiumperfringens,
DetectionofClostridium
perfringensalphatoxin—
isolatedintheintestine
Viralinfection
MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,focal
broncho‐pneumonia,
hemorrhagicgastroenteritis
Caninedistempervirus—
detectedinthelung,
liver,andbrain
Pasteurellaspp.—detectedin
thelung
Viralandbacterialco‐
infection
MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,
hemorrhagicgastroenteritis
NovirusesdetectedNobacteriadetectedUndetermined

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MAcuterespiratory
insufficiency
Visceralcongestion,
pulmonaryedema,severe
broncho‐pneumonia
NovirusesdetectedNobacteriadetectedUndetermined
MAcuterespiratory
insufficiency
Visceralcongestion,
hemorrhagicenteritis
Canineparvovirustype2a
(wildtype)—detectedin
theheart,spleen,and
intestine
NobacteriadetectedViralinfection
3
M
Sialorrhea,unilateraleye
swelling,muscle
stiffness,asingle
episodeofvomiting
Visceralcongestion,
pulmonaryedema,
hemorrhagicenteritis
Novirusdetected
E.coli;Clostridiumperfringens
DetectionofClostridium
perfringensalphatoxin—
detectedintheintestineand
lung
Bacterialinfection
MNeurologicalsymptoms
Visceralcongestion,bi‐
lateralpneumonia,
pulmonaryedema
segmentalcatarrhal
enteritis
Rotavirus(detectedin
theintestine)
E.coli—isolatedinthe
intestine
Undetermined:
severepneumonia
duetounexplainedcauses
FAcutegastrointestinal
symptoms
Visceralcongestion,
hemorrhagicenteritis,focal
pneumonia
Canineparvovirustype2a,
(wildtype)—detectedin
thelung,liver,and
intestine;Adenovirus—
detectedinintestine
Clostridiumperfringens
DetectionofClostridium
perfringensalphatoxin—
isolatedintheintestine
Viralinfection
FVomiting
Visceralcongestion,
hemorrhagicenteritis,focal
pneumonia
Canineparvovirustype2a
(wildtype)—detectedin
thelung,liver,heart,
andintestine
E.coli—isolatedinintestine
andlung
Viralandbacterialco‐
infection
MVomiting
Visceralcongestion,
pulmonarycongestion,
enteritis,abdominal,
thoracicandpericardial
effusion,multifocal
pulmonaryhemorrhage
NovirusdetectedE.coli—isolatedinintestine
Undetermined:
insufficientfindings
toexplaindeath
4FAcuterespiratory
insufficiency
Multifocalhemorrhage,
abdominal,thoracicand
pericardialeffusion,
hemorrhagicenteritis
Novirusdetected
E.coli—detectedintheliver,
lung,andintestineClostridium
perfringensDetectionof
Clostridiumperfringensalpha
toxin—detectedinthe
intestineandlung
Bacterialinfection

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5
MAcuterespiratory
insufficiency
Pulmonarycongestion,
segmentalcatarrhal
enteritis
Canineparvovirustype2a
(wildtype)—detectedin
lung,liver,andintestine
E.coli—isolatedinthe
intestineViralinfection
FAsingleepisodeof
diarrhea
Pulmonarycongestion,
pulmonaryedema,
segmentalhemorrhagic
enteritis
Canineparvovirustype2a
(wildtype)—detectedin
lung,liver,intestine,and
spleen
Adenovirus—detectedin
thelung
NobacteriadetectedViralinfection
6
FLackofappetite
Thoraciceffusion,visceral
congestion,multifocal
hemorrhage,severe
hemorrhagicenteritis
Canineparvovirustype2a
(vaccinal)and2c(wild
type)—detectedinlung,
liver,intestine,and
spleen
Caninedistempervirus‐
detectedinlung
Adenovirus—detectedin
lung
NobacteriadetectedViralinfection
FLackofappetiteand
feverfor12h
Multifocalhepaticnecrosis,
hemorrhagicenteritis
Canineparvovirustype2b
(wildtype)—detectedin
thelung,liver,brain,
andintestine
E.coli.Clostridiumperfringens,
DetectionofClostridium
perfringensalphatoxin
(isolatedinlung,liver,and
intestine)
Viralandbacterialinfection
FSingleepisodeof
diarrhea
Congestionofthespleen,
abdominaleffusionNovirusesdetectedNobacteriadetected
Undetermined:
insufficientfindings
toexplaindeath

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4.Discussion
Inhumanforensicpathology,theautopsyforcasesofSUIDareprimarilyperformedaccording
tothe“KennedyReport”[11].Thisprotocolandthepublishedinternationalguidelinesadvocatea
multidisciplinaryapproachtoinvestigationsofallcasesofSUID,whichshouldbebasednotonlyon
thefindingsofthepost‐mortemmacroscopicexamination,butalsoonabroadrangeofancillary
investigations,suchasbacteriologicalandvirologicalanalyses.Althoughabroadrangeoftestshave
beenproposedincasesofSUIDinhumanforensicmedicine,inthepresentstudy,wefocusedonthe
pathologicalandpost‐mortemmicrobiologyfindingsincasesofsuddendeathinyoungdogs.The
resultsofthisstudyshowalowfrequencyofsuddendeathsinyoungdogs,accountingfor14.48%of
thetotalobserveddeaths.Furthermore,theChi‐squaredtestshowedasignificantdifferenceinthe
frequenciesofEDandSUDamongtheassessedagegroups(p<0.05).Inparticular,thehighest
frequencyofsuddendeathwasobservedindogsyoungerthan4weeksold.Incontrast,thehighest
frequencyofEDwasobservedinanimalsinGroups2–5.Thisdifferencecouldbeduetothe
immaturityoftheimmunesystemofpuppiesyoungerthan6–12weeksofage[24].Indeed,the
endotheliochorialplacentationofthisspeciesisrelativelyimpenetrabletothetransferofmaternal
immunoglobulin[24].Thus,theimmuneprotectionofthepuppiesduringthefirstweeksoflife
dependsontheingestionofmaternalcolostrumantibodies(MCA)[24].Intheabsenceofthepassive
transferofMCA,newbornpuppiesareonlyabletodevelopanimmuneresponsetoantigensat2–3
weeksofage.Therefore,anydelayincolostrumintakeorreductionofcolostrumingestionleadstoa
reductionintheimmuneprotectionoftheanimals[24–26].Undertheseconditions,virusesorbacteria
canreplicateandspreadquickly,leadingtothedeathofthepuppies,withoutthedevelopmentof
characteristicsymptoms.Furthermore,congenitalmalformationormaternalmalnutritioncouldbe
consideredadditionalcausesofsuddendeathinthisagerange.Incontrast,after2–3weeksofage,
theimmunesystemofpuppies,althoughimmature,isabletodevelopamildimmuneresponse
againstpathogens,avoidingtherapidspreadofthepathogensandallowingthedevelopmentof
characteristicsymptomsofthepathology.Inaddition,inourstudy,themostfrequentlyobserved
grossinjuriesincasesofsuddendeathwerehaemorrhagicgastroenteritisin12outof21cases,
pneumoniainsevenoutof21cases,andcatarrhalenteritisinfiveoutof21cases.Pulmonaryoedema
ormultiorgancongestionwerealsoobservedin19outof21cases.Intra‐abdominalandrespiratory
lesionshavebeenpreviouslyreportedintheliteratureastwoimportantcausesofsuddenand
unexpectedinfantdeathinhumanforensicpathology[27].Indeed,respiratorytractlesions,although
mild,caneasilyleadtoseriouscomplicationsandsuddendeathofthesubjects[27].Similarly,acute
gastroentericlesionscancauseseveredehydrationandserumelectrolytedisturbance,whichhave
thepotentialtocausesuddenandunexpecteddeathinchildren[27].Overall,inourstudy,positive
microbiologicalresultswereobservedin18outof21cases.However,asfrequentlyhighlightedin
thehumanliterature,theisolationofpathogensincasesofsuddendeathdoesnotnecessarilyimply
acorrelationbetweenthosepathogensandthedeath.Inparticular,thiscorrelationmustbeconfirmed
bytheobservationofsevereandspecificinjuriesduringtheanatomopathologicalexamination[6].
Therefore,afterthereviewofthenecropsyfindings,detectedpathogenswereconsideredthemain
causeofdeathinonly14outof21cases,whileintheremainingsevencases,themicrobiological
resultsdidnotexplaintheinjuriesobservedduringthenecropsy.Thenegativefindingsobservedin
ourstudycouldsuggest:(1)anon‐infectiouscauseofdeathoftheassessedanimals;or(2)adeath
duetovirusesorotherpathogensnotdetectedbythevirologicalpanelinuseinthisstudy.Indeed,
thereareawiderangeofvirusesthatarepotentiallypathogenicinyoungdogs,includingbothDNA
andRNAviruses.However,ourvirologicalpanelwaslimitedtothedetectionofthefollowingfive
specificviruses:canineparvovirus,caninecoronavirus,canineadenovirus,canineherpesvirusandcanine
distempervirus.Withregardtothepositiveresults,viralinfectionduetocanineparvovirustype2(wild
type)wasthemostcommoncauseofdeathobservedinourstudy.Overall,CPV‐2isacausativeagent
ofacutegastroenteritisandmyocarditis[17,18].Furthermore,itisreportedintheliteraturetobeone
ofthemostcommonandimportantcausesofmorbidityandmortalityinyoungdogs[17,18].Usually,
theclinicalsymptomsoftheinfectionareasfollows:anorexia,depression,lethargyandfever,

Animals2020,10,113410of12
followedbyvomitinganddiarrhea[17,18,28].However,itisalsoreportedtobeacauseofsudden
cardiacdeathinpuppiesbetween4and8weeksofage[17,29,30].Interestingly,wealsoobservedtwo
casesofsuddendeathduetoCaninedistempervirusandPasteurellaspp.co‐infection.Caninedistemper
virusisamemberofthegenusMorbillivirus,whichcancausealargevarietyofdisordersindogs
includingrhinitis,pneumonia,demyelinatingleukoencephalitis,necrotizingbronchiolitisand
enteritis[31].Caninedistempervirusisnotreportedintheveterinaryliteratureasacauseofsudden
deathinanimals.However,Caninedistemperviruscausesimmunosuppressionbytargetingcellsthat
expresstheCD150protein(signalinglymphocyteactivationmolecule(SLAM))[31].Previousstudies
showedthatthisimmunosuppressionfavorssecondaryinfectionscausedbypathogens,suchas
BordetellabronchisepticaorC.piliforme[32].Therefore,itispossibletosupposethatopportunistic
pathogenscouldcomplicateasub‐clinicalCaninedistempervirusinfection,replicatingandspreading
quickly,andleadingtothedeathofthepuppieswithoutthedevelopmentofcharacteristicsymptoms.
Withregardstothebacteriologicalexamination,themostcommonisolatedbacteriawereC.
perfringenstypeA(6/19)andE.coli(6/19).However,theywereconsideredthecauseofdeathinonly
twocases.Indeed,C.perfrigensandE.coliareconsiderednormalcomponentsofcanineintestinalflora
[33,34].Similarly,thealphatoxingeneofC.perfringensmaybefoundinasymptomaticdogsaspart
ofthenormalintestinalmicroflora[33].However,insomecases,E.colicancausepleuro‐pneumonitis
[35],gastroenteritis[36],urogenitalinfections,cholangitis,cholangiohepatitisandsepticaemia[37]in
bothhumansandotheranimals.Similarly,C.perfringenstypeAhasbeenassociatedwithgastro‐
entericdisease,suchashaemorrhagicenteritisindogsandabomasitisinruminants[38–40].
Furthermore,thisbacteriumhasbeenreportedintheliteratureasacauseofsuddenandunexpected
deathindogs[39].Unfortunately,nospecifictestforthediagnosisofenteritisduetoC.perfringensis
describedintheliterature[40].Thus,generally,theclinicalsigns,thepathologicalfindings,the
microbiologicalanalysis,andtheabsenceofotherpathogensmustbeexaminedbeforeconfirming
thediagnosis[40].Inourcase,themultisitelocationsofthepathogens,theabsenceofotherviruses
orbacteriaandthespecificanatomopathologicalfindingsofhaemorrhagicenteritissupportedthe
diagnosisofenteritisduetoC.perfringensandE.coliasthefinalcauseofdeath.
Finally,thisstudyallowedthedetectionofawiderangeofpathogensthat,afterthereviewof
thenecropsyandmicrobiologicalreports,werenotconsideredthemaincauseofdeathoftheanimals,
suchasCanineAdenovirus,Rotavirus,Streptococcussanguinis,Streptococcusdysgalactiaeand,insome
cases,E.coliandC.perfringens.Therefore,furtherstudieswillbeneededtoevaluatethepossible
contributionsofthesepathogenstocasesofsuddenandunexpecteddeathinyoungdogs.
5.Conclusions
Suddendeathisanuncommoncauseofdeathinyoungdogs.However,thehighfrequenciesof
virusesandbacteriadetectedinourstudyhighlightstheimportanceofperformingcomplete
bacteriologicalandvirologicalanalysesinallcasesofsuddendeathinyoungdogs.Theresultsofthis
studysuggestthatourPCRpanelcombinedwithabacteriologicalanalysiscouldfacilitatetherapid
detectionandtype‐specificidentificationofthepathogeniccausesorco‐factorsofdeathinmostcases
ofsuddendeathinyoungdogs.Finally,theseresultscouldprovideavaluableepidemiologicaltool
fortheinvestigationofsuddendeathinyoungdogs.
AuthorContributions:Theindividualcontributionsinthepresentstudywereasfollows:conceptualizationand
methodologyG.P.,G.F.,L.C.;validationF.A.,L.V.,V.I.;investigationG.P.,L.C.;datacurationG.P.;writing—
originaldraftpreparationG.P.;projectadministrationG.F.;fundingacquisitionG.F.Allauthorshavereadand
agreedtothefinalversionofthemanuscript.
Funding:Thisresearchdidnotreceiveanyspecificgrantfromfundingagenciesinthepublic,commercial,or
not‐for‐profitsectors.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.


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