Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

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

Veterinary

Microbiology

Contribution

of

ﬂagella

and

motility

to

gut

colonisation

and

pathogenicity

of

Salmonella

Enteritidis

in

the

chicken

Fernanda

de

Oliveira

Barbosa

a

_,

_Oliveiro

_Caetano

_de

_Freitas

_Neto

b,∗

_,

Diego

Felipe

Alves

Batista

a

_,

_Adriana

_Maria

_de

_Almeida

a

_,

_Marcela

_da

_Silva

_Rubio

a

_,

Lucas

Bocchini

Rodrigues

Alves

a

_,

_Rosemeire

_de

_Oliveira

_Vasconcelos

a

_,

Paul

Andrew

Barrow

c

_,

_Angelo

_Berchieri

_Junior

a

a_Departamento_de_Patologia_{Veterinária,}_Faculdade_de_Ciências_Agrárias_e_{Veterinárias,}_Universidade_Estadual_Paulista,_Jaboticabal,_São

Paulo,Brasil

b_Departamento_de_Ciências_{Veterinárias,}_Universidade_Federal_da_Paraíba,_Areia,_Paraiba,_Brasil

c_School_of_Veterinary_Medicine_and_Science,_University_of_Nottingham,_Sutton_Bonington_campus,_{Leicestershire,}_United_Kingdom

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Articlehistory:

Received16August2016

Accepted20January2017

Availableonline7June2017

AssociateEditor:RoxanePiazza

Keywords: Mutation Flagellum Virulencefactors Poultry Gutcolonisation

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SalmonellaEnteritidiscausesfowlparatyphoidinpoultryandisfrequentlyassociatedto

out-breaksoffood-bornediseasesinhumans.Theroleofﬂagellaandﬂagella-mediatedmotility

intohost-pathogeninterplayisnotfullyunderstoodandrequiresfurtherinvestigation.In

thisstudy,one-day-oldchickenswerechallengedorallywithawild-typestrainSalmonella

Enteritidis,anon-motilebutfullyflagellated(SEmotB)ornon-flagellated(SEfliC)strainto

evaluatetheirabilitytocolonisetheintestineandspreadsystemicallyandalsoofeliciting

grossandhistopathologicalchanges.SEmotBandSEﬂiCwererecoveredinsigniﬁcantly

lowernumbersfromcaecalcontentsincomparisonwithSalmonellaEnteritidisatearlystages

ofinfection(3and5dpi).TheSEmotBstrain,whichsynthesisesparalysedﬂagella,showed

poorerintestinalcolonisationabilitythanthenon-ﬂagellatedSEﬂiC.Histopathological

analysesdemonstratedthattheﬂagellatedstrainsinducedmoreintenselymphoidreactivityin

liver,ileumandcaeca.Thus,inthepresentstudytheﬂagellarstructureandmotilityseemed

toplayaroleintheearlystagesoftheintestinalcolonisationbySalmonellaEnteritidisinthe

chicken.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Corresponding}_author_at:_Department_of_Veterinary_Sciences,_Federal_University_of_Paraiba,_Paraiba_58051900,_Brazil.

E-mail:oliveirocaetano@yahoo.com.br(O.C.FreitasNeto).

http://dx.doi.org/10.1016/j.bjm.2017.01.012

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Introduction

Salmonellaentericasubsp.entericaserovarEnteritidis(SE)isa

broad-hostrangemicro-organismwhichposesathreattoboth

publicandanimalhealth.1_It_causes_fowl_paratyphoid,_which_is

oftenassociatedwithextensivegutcolonisationandbacterial

sheddinginthefaeces.2,3_SE_is_one_of_the_commonest_serovars

relatedtoworldwidefood-borneoutbreaks.4

Following oral infection ﬂagellated strains of Salmonella

spp.colonisetheintestinesandﬂagellin,the mainﬂagellar

protein,isrecognisedthroughToll-likereceptor(TLR)-5

lead-ingtoactivationofapro-inﬂammatoryresponseandrelease

ofthecytokinesnecessarytoinitiatetheinnateandadaptive

immuneresponses.5_The_intense_local_{inﬂammation}_triggered

duringdiseasehelpsrestrictthebacteriatotheintestineand

helpstocontrolthesystemicinfection.6

Despiteactivatingtheinnateimmunity,possessionof

ﬂa-gellaisanimportantvirulencetraitwhichmediatesbacterial

attachment and invasion.7,8 _In _addition, _{ﬂagella-mediated}

motilityhasalsobeenconsideredasavirulencedeterminant

forgut-associated Salmonella. Thus, a non-motileSE strain

showedreducedabilitytoattachtocellsincomparisontothe

parentalstrain.9

The correlation between ﬂagella and ﬂagella-mediated

motilityand whether ornottheycontributeindependently

toSalmonellapathogenesisisunknown.Toinvestigatethis,

non-motilebut fully ﬂagellated (SEmotB) and non-motile

andnon-ﬂagellated(SEﬂiC)mutantstrainswereconstructed

and the roles ofﬂagella and ﬂagella-mediated motility on

intestinalcolonisationandsystemicinvasionofchickenswere

assessed.

Materials

and

methods

Bacteria

Thisstudyusedthespontaneousnalidixicacidresistantstrain

P125109(SE).Theparentstrain wasisolatedfromacaseof

food-poisoninginhumansandisvirulentforyoungchickens

andcapableofcontaminatingeggswheninoculatedinlaying

hens.10_All_bacteria_were_cultured_in_lysogeny_broth_(LB_–

Bec-tonDickinson,Sparks,Maryland,USA)at37◦Cfor24hat150

revolutionspermin(rpm).11

Mutantconstruction

Twomutantstrains,SEﬂiCandSEmotB,wereconstructed

usingtheLambda-redmethod12 _and _transduction_with_the

phageP22wasusedtotransferthemutationtoacleangenetic

background.PutativemutantswereselectedinLysogenyagar

(LA – DifcoTM_, _Detroit, _Michigan, _US) _containing ₂₀_␮g/mL

chloramphenicolandconﬁrmedbypolymerasechainreaction

(PCR).Afterselection, the chloramphenicol-resistancegene

waseliminatedbyusingahelperplasmidexpressingtheFLP

recombinase(pCP20),whichactsonthedirectlyrepeatedFRT

(FLPrecognitiontarget)sitesﬂankingtheresistancegene.

Spe-ciﬁcprimersweredesignedthroughPrimerBlasttool13_and_are

availableinTable1.

Flagellaandﬂagella-mediatedmotilitydetection

SE,SEmotBand SEﬂiCswimmingmotilitywasdetected

by propagation on semi-solid agar (SSA), after inoculation

onto the surface of semi-solid plates consisting of 0.9%

heart infusion broth (Oxoid, Basingstoke, Hampshire, UK)

and 0.25%LA(Difco,Detroit,Michigan, US),after24h

incu-bation at 28◦C assessed by bacterial spread through the

soft agar. Flagella expression was additionally conﬁrmed

throughserum-agglutinationusingspeciﬁcanti-H:g,m

anti-bodies(Remel,Dartford,Kent,UK).

Chickens

One hundred and seventy one one-day-old male chickens

fromacommerciallineofegglayerwereusedinthetwo

exper-iments.Birdswerehousedinacclimatisedroomsandreceived

waterandfeedadlibitum.Onarrival,samplesoffaecesinthe

transportcardboard boxeswere collectedand processedto

excludeinfectionwithSalmonellaspp.11_In_each_experiment

birdsininfectedgroupsreceived1×109_colony_forming_units

(CFU)ofSE,SEmotBorSEﬂiC,respectively,intothecrop

usingoralgavageneedles.Experimentswereapprovedbythe

institutionalethicalcommittee(Process1.353/15;approvedon

03March2015).

Experiment1–mortality,clinicalsignsandfaecal shedding

Forty-ﬁve chickens were distributed randomly into three

groups of15 animals and then infected. Birds of infected

groupsreceived1×109_CFU_of_SE,_SE_motB_or_SE_ﬂiC,

respec-tively, into the cropasabove.Birdswere observed forfour

weeks.Mortalityandotherclinicalsignswererecordeddaily

and bacterial sheddinginfaeceswas monitoredbycloacal

swabstwiceaweek.11

Experiment2–localandsystemicinfectionand pathologicalchanges

Onehundredandﬁvechickensweredistributedrandomlyinto

threegroupsof35animalsandtheninfected.Birdsininfected

groupswereinfectedorallyinoculatedwith1×109_CFU_of_SE,

SEmotBorSEﬂiC,respectively.Afourthgroupof21chicks

waskeptastheuninfectedcontrolforhistopathology.Birdsof

uninfectedcontrolgroupwerealsomock-infectedwith0.2mL

ofsterilelysogenybroth(LB–BectonDickinson,Sparks,

Mary-land,USA).At2,3,5,7,14,21and28dayspost-infection(dpi),

ﬁvebirdsfromeachinfectedgroupwereeuthanasedby

cervi-caldislocationandsamplesofspleen,liverandcaecalcontent

collectedforbacterialenumeration.11_Gross_pathologies_were

alsorecorded.

At the time points above samplesof liver, caecum and

ileum were collected from the same infected chicks and

also from three non-infected animals forhistopathology.15

Sampleswereformalin-ﬁxedandparafﬁn-embedded.Tissues

weresectionedat4-␮mthickness,stainedwithhaematoxylin

andeosinandobservedbylightmicroscopy.Lesionswere

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Table1–PrimersequencesusedtoconstructtheSEﬂiCandSEmotBmutantstrains.

Primer Sequence Reference

C1 5-ttatacgcaaggcgacaagg-3 12

C2 5-gatcttccgtcacaggtagg-3 12

motBF 5-tgccgtggaatttggtcgta-3 Thisstudy

motBR 5-atccagagttgccgacagtg-3 Thisstudy

motB75F 5-atgaaaaatcaggctcatcccattgtcgtcgtaaaacgccgcaggcacaaaccgcacggcggcggggcgcgtgtaggctggagctgcttc-3 Thisstudy motB75R 5-tcacctcggttccgcttttggcgatgtgggtacgcttgccggcggggctgccgcaggctgttgtaatacacttaccatatgaatatcctccttag-3 Thisstudy

ﬂiCctrF 5_{-gttatcggcaatctggaagc-3} ₁₄

ﬂiCctrR 5_{-ggtgacaaaggcaggttcag-3} ₁₄

ﬂiC50F 5-gatacaagggttacggtgagaaaccgtgggcaacagcccaataagtgtaggctggagctgcttc-3 14 ﬂiC50R 5-ctttcgctgccttgattgtgtaccacgtgtcggtgaatcaatcgccggacatatgaatatcctccttag-3 14 Longprimerswereusedforamplifyingantibioticcassettes.Shorterprimerswereusedforverifyingcassetteinsertion.

Statisticalanalysis

Dataonmortalityandfaecalsheddingwerecomparedby

chi-squaretest.17_Statistical_differences_amongst_viable_bacteria

numbersrecoveredfromcaecalcontents,liversandspleens

were determined using Tukey’s test.6 _Statistical _tests _were

performedusingGraphPadPrismversion6.00 forWindows

(GraphPadSoftware,LaJolla,California,USA).

Results

Mutagenesis–ﬂagellaandﬂagella-mediatedmotility assessment

DeletionofﬂiCandmotBgenesfromtheSEchromosomewas

ﬁrstconﬁrmedbyPCR.Themutantstrainsshowed

impair-mentintheirabilitytospreadthroughouttheSSAafter24h

incubationandonlyasmallhalo,nearly6-mmdiameter,was

noticeableinthecentreoftheagar.Bycontrast,SEwasable

tocoverthewholesemi-solidsurfaceafter24hofincubation.

Theserumagglutinationtesttargetingtheﬂagellarantigens

(H:g,m)waspositiveforSEandSEmotBstrainsandnegative

forSEﬂiC.

Experiment1–mortalityandfaecalshedding

Clinicalmanifestationsbeganat4dpiinallinfectedchickens

inwhichsomnolence,closedeyesandpersistentdiarrhoea(up

to13dpi)containingsmearsofbloodwereobserved.SEand

SEﬂiCinfectionsproduced13%mortality(n=2/15infected)

whereas nomortalityoccurred amongst SEmotB-infected

chickens.Despite this,nostatisticalsigniﬁcancewasfound

betweenthemortalityrates(p>0.05).Additionally,the

num-berofpositivecloacalswabsfromwhichtheinoculatedstrain

wasrecoveredwasverysimilaramongsttheanimalsinfected

withSE(92.5%),SEmotB(87.5%)andSEﬂiC(93.3%),andit

wasnotstatisticallysigniﬁcant(p>0.05).

Experiment2

Caecalcolonisationandsystemicinvasion

Theresultsofbacterial enumerationinlivers, spleensand

caecalcontentsareshowninFig.1.Therewasnostatistically

signiﬁcantdifferencebetweenthebacterialnumbersincaecal

content (p=0.7225), liver (p=0.5618) and spleen (p=0.5294)

at2dpi.SEcolonisedthecaecalcontentsinhighernumbers

early (3, 5 and 7 dpi)in infection (p<0.05). However, from

14dpi onwardthe bacterial counts of all strains incaecal

contentsdecreasedtosimilarnumbers(p=0.6257).Bacterial

recoveryfromliversandspleenswasverysimilarforallthree

strains. SEreachedthespleensinhighernumbers at3dpi

(p<0.05) but at5 dpi onward all strains showed a similar

behaviour (p=0.1880). The bacterial numbers in the livers

werelow(103_CFU/g)_throughout_the_experiment_for_all_three

strainsandnostatisticalsigniﬁcancewasfound(p=0.3513).

Pathologicalchanges

Nogrosspathology wasobservedinanyinfectedanimalat

2dpi. From 3dpi, mild hepatosplenomegaly and mild

hae-morrhagic enteritis were observedin SE-infectedchickens,

whereas no noticeable changes occurred in the intestines

of SE ﬂiC- and SE motB-infected chickens. The greatest

changes, however, were noticed at 7dpi when congestive

hepatosplenomegaly andthickened intestinalmucosawere

noticeableinallnecropsiedanimals.From14to28dpigross

pathologiesbecamemildbutpresentinallinfectedanimals.

Themostseverehistopathologicalchangeswereobserved

in the liver, ileum and caeca of SE-infected chickens. SE

induced hepatocyte degeneration and lymphoid reactivity

from 2dpi,butat7dpi,theformer becamesevereand

diff-used and the latter moderate and mostly surrounding the

portaltriadsandperivascularareas.Duringthissamespanof

time(2–7dpi)SEmotBinducedmilderhepatocyte

degenera-tionandmoderatelymphoidreactivitysurroundingtheportal

triadsandperivascularareaswhereasSEﬂiCprovokedmild

fociofnecrosiswithmildadjacentinﬁltrationofmononuclear

cellsinthehepaticparenchyma.At14,21and28dpimild

hep-atocytedegenerationwithlymphoidreactivityatparenchyma

wasseeninliversinallinfectedanimals.

InthegutSEelicitedmoderatemultifocallymphocyte

inﬁl-tration inthe ileallamina propriamucosa from 2to7dpi.

At5dpi SEmotBelicitedmild multifocallymphocyte

inﬁl-trationintheileallaminapropria(Fig.2).MeanwhileinSE

ﬂiC-infectedchickensthisalterationwasobservedlater,at

7dpi.From14dpionwards,lymphocyteinﬁltrationintheileal

laminapropriabecamemoderateinbirdsinfectedwithboth

mutant strains. By contrast,SE causeddiffuseand

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Caecal contents

Spleen

Liver

1 3 b b b b a a a b b 5 7

Days post infection

Days post infection _{Days post infection}

SE ΔfliC SE ΔmotB SE 15 10 5 0 6 4 2 0 6 4 2 0 Log 10 CFU/g Log 10 CFU/g Log 10 of CFU/g 14 21 28 1 3 5 ₇ ₁₄ ₂₁ ₂₈ 1 3 a ab b 5 7 14 21 28

Fig.1–Bacterialcounts(log10CFU/g)inlivers,spleensandcaecalcontentscollectedfromone-day-oldchicksinfectedwith

SE,SEﬂiCandSEmotB.DifferentlettersontheplotsmeantherewasstatisticalsigniﬁcancebyTukey’stest(p<0.05) betweendistincttreatmentsbyday.

experiments.TheSEﬂiCand SEmotBstrainscausedthe

samelesions,butat3and5dpimildlymphocyteinﬁltration

inthecaecallaminapropriawasobserved.Mononuclearcell

inﬁltrationinlaminapropriainadditiontovillusfusionand

submucosaloedemabecamemildandsimilarinallinfected

birdsafter21dpi.

Discussion

Flagellaandﬂagella-mediatedmotilityareconsidered

impor-tant factors for salmonellosis.18 _Their _contribution _to _S.

Enteritidis(SE) pathogenicityinpoultryhasbeenevaluated

inseparatestudies,8,19,20_but_the_role_of_ﬂagella_as_opposed_to

motilitystillrequiresfurtherinvestigation.Toshedlighton

thissubject,thepresentstudycomparedtheinfectionbiology

ofthemotileandfullyﬂagellatedSEstrainP125109andits

derivativemutantstrains,onenon-motileandnon-ﬂagellated

(SEﬂiC)andothernon-motilebutﬂagellated(SEmotB)using

one-day-oldmalechickensasthemodel.

Overthe4-weekexperiment1bacterialrecoveryfrom

fae-ceswassimilarforallstrainsindependentonthephenotype.

Inagreementwiththisresult,apreviousreportshowedthat

theinfectionofchicksbyawild-typeSEandanon-motile

ﬂag-ellatedresultedinasimilardegreeoffaecalexcretion.20_These

ﬁndings,combinedwiththeabsenceofsigniﬁcantmortality

inthepresentstudy,showthatneithertheabsenceofﬂagella

noritsrelatedmotilityalterthefaecalexcretionabilityofSE

inchickens.

Althoughthemutationsintroducedinto theSE

chromo-somedidnotimpairbacterialsheddingbyfaeces,theability

tocolonisethecaecaearly wasalteredsincethe countsof

SEﬂiCandSEmotBincaecalcontentsat3and5dpiwere

signiﬁcantlylower.Previousstudiesusingchickeninfection,

chickengutexplantsorculturedepithelialcellsinfectedwith

non-motile strainsofSalmonella, alsoreported the reduced

abilityofthesemutantstrainstocolonise/adheretothecells

whencomparingtothewild typeﬂagellated strains,inthe

earlystagesofcolonisation.7,19,21_Taken_together_these_results

suggestthatﬂagellaandﬂagella-mediatedmotilitywouldplay

importantroles early (upto5dpi),but not later,duringSE

infectioninchickens.Thisconclusionisalsosupportedbythe

factthatat7dpibothmutantstrainsstartedtocause

intesti-nalhistopathologicalchangessimilartothoseinducedbythe

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Fig.2–Transversesectionsofileacollectedat5dpifromchicksinfectedat1dayoflifewithSE,SEmotBorSEfliC.(A) Healthyileumcollectedfromanuninfectedbirdshowingnomononuclearinfiltration(arrow);(B)presenceofmild mononuclearinflammatoryinfiltrateinthemucosa(arrow)ofaSEmotB-infectedchick;(C)nodetectablechangesinileal mucosa(arrow)ofaSEfliC-infectedchick;(D)presenceofseveremononuclearinflammatoryinfiltrate(arrow)infusedand shortenedvilliintheilealmucosaofaSE-infectedchick.Haematoxylinandeosinstaining.Scalebars:100␮M(small images)and40␮M(largeimages).

Ithas been hypothesised that recognition ofﬂagellated

Salmonella strains through intestinal TLR5 leads to

activa-tion of pro-inﬂammatory response which in turn helps to

restrictthebacteriatotheintestineandtopreventsystemic

infection.5,7 _However,_in_the_present_study,_at₃_dpi_the_wild

typeSEstrain wasrecovered fromspleeninhigher counts

than thenon-ﬂagellated SEﬂiC.Very similarresultswere

reportedinratsinfectedwithﬂagellatedand

non-ﬂagellated-SEstrains.20_It_seems_that_the_absence_of_ﬂagella_in_SE_was_in

factdisadvantageousinestablishingsystemicinfection.

Fur-therstudiesmustbecarriedoutinordertobettercharacterise

theimmunologicalbasesoftheinfectionbythesestrainsand

toassesswhetherornotthelowersystemiccolonisationof

SEﬂiCisaconsequenceofitsreducedinvasionabilitydueto

theabsenceofmotility.9

Interestingly,SEﬂiC,butnotSEmotB,showedimproved

ability to colonise the caeca at 7dpi. Similar results were

previouslyreported forSalmonella Typhimurium (STM)in a

murine ligated-loop invasion assay. It was postulated that

theextracellularelectrostaticrepulsionproducedaroundthe

paralysedﬂagellapreventedthecontactbetweenbacteriaand

intestinalcells,thusaffectingthegutcolonisation.22_This

phe-nomenon,designatedassterichindrance,couldalsobethe

possibleexplanationforthelongerlowerlevelcaecalrecovery

ofSE motB comparedtoSE ﬂiCobservedin the present

study.

Thewild-typestraininduced,attheearlystagesof

infec-tion,moreseverehepaticlesions.AccordingtoXiaoetal.,23

ﬂagellaexpressionisinhibitedintheliveralthoughaminimal

amountofflagellinreleasedbyflagellatedstrainsissufficient

to stimulatethe immune systemvia TLR5recognition and

inducesubsequentfunctionabnormalityanddamagetothe

liver.SEﬂiCdoesnotproduceﬂagellinwhichisthoughtto

bethereasonbywhichonlymildhepaticlesionswere

pro-ducedbythisstrainearlyininfection.InthisstudySEmotB

inducedinﬂammatoryinﬁltrationinilealandcaecamucosae.

ThisresultagreeswiththatshownbyXiaoetal.23_in_which

paralysedﬂagellawereassociatedwithasigniﬁcantreduction

ininvitroinvasivenessalthoughpresumablystillabletosignal

throughTLR5.

Datageneratedinthisstudyshowedthatthelackofeither

ﬂagella or ﬂagella-mediatedmotility impairs SE

pathogen-icity in young chickens, chieﬂy in the intestine and early

duringinfection.Theparalysedﬂagellaalsoappearedtobe

more detrimentalthan the complete absence ofﬂagella,a

factdemonstratedpreviouslyinepithelialcells.9_These_results

implythatmotilityinSalmonellacontributestotheearlystages

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Conﬂicts

of

interest

Thereisnoconﬂictofinterest.

Acknowledgments

This work was supported by São Paulo Research

Founda-tion(FAPESP)[grantnumbers2014/02014-1(F.O.Barbosa)and

2013/26127-7 (A. Berchieri Junior)] and National Council of

TechnologicalandScientiﬁcDevelopment(CNPq).

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